Hey guys and welcome back to my latest video. In today's video I am partnering with Agogear who sent me these amazing looking scrubs and I'm so excited to do a review video for you guys and stay tuned to the end because there will be a giveaway and a promo code. So I have to be honest with you guys, I have worn a lot of different brands of scrubs and Agogear scrubs has to be by far the best quality fabric that I have ever come in contact with. It's like the best of both worlds. So I feel like on the scrub spectrum you have scrubs that are like more starchy and not very stretchy to like athletic stretchy kind of under armor, so to speak type of material. And I feel like these are like the best of both worlds because they're stretchy, but at the same time they are like durable and feel very like, how do I describe it? When I wore these scrubs to my first shift and to try them out, I had several of my coworkers come up to me and be like, did you get new scrubs? Did you get new scrubs? Which is weird because like we all wear lots of different brands of scrubs and like, I don't know. I didn't think it was that noticeable but they're like, wow, those scrubs are really nice. And one of my friends was like feeling the fabric and she was like, wow, these scrubs feel like very durable. I'm like, I know they're like stretchy but at the same time I don't feel like after I wash them four times they're just gonna like disintegrate and fall apart. I think my favorite feature of these scrubs and at first I was like, this is kind of weird. So I put these scrubs on, it's like 5.30 in the morning I'm getting ready for and I'm like, is this the collar on the scrubs? And I'm like trying to like, I'm like, do I like pop it over on my scrubs? And I was like, that's interesting to have like this little neck part. But then I realized when I got to work it's actually meant for your stethoscope. At least that's what I'm assuming it's meant for. And I personally hate when my stethoscope like rubs against my neck. It like irritates me so bad and throughout my shifts I'm constantly like adjusting it and it's sliding and it's annoying. But with this you have this little like barrier so that way it like, it doesn't slide but it also like protects your neck so it's just not irritating. So I'm like, this is an awesome feature that I have never seen on any scrubs before and I didn't realize it was something that I like needed with my scrubs but they haven't. Another cool feature I really love is this little loop, I don't know what you wanna call it but you can like put pens in there or also like you could put your badge on there and that's where I put my badges on this little loop because if I put it up here it tends to like slide down and by the end of the day it's like right here and it's like pulling my scrubs down and people are always like, hey Ashley like pull your badge up and it just happens and I can't help it because it just doesn't stay very well but here you can have your badge like still right in plain sight and it won't slide which I think is awesome. The other great part of these scrubs is they're water repellent and they're also antimicrobial which you know how many bugs that we were exposed to when we were just a few shifts ago.   So of course we could not do a review video without doing a giveaway. So Eagle Gear scrubs was just so generous to do a giveaway of one of their pairs of scrubs and you can choose the style and the color and the size and so it'll be perfect for you and your needs so all of the giveaway instructions will be down below and they're also offering a promo code which I will leave right here for if you wanna save 25% off your purchase which is huge 25% off is a legit deal. I feel like most times people wanna do like 10, 15% off which is awesome but 25 is even better. So if you're interested in checking out Eagle Gear scrubs and using the promo code go to the link down below check out the giveaway instructions down below and I am so excited that I gotta share these scrubs with you if you get a pair which you take a picture and tag me in your post or send it to me or whatever because I just always love hearing your opinions on these products because obviously I only promote products that I know you guys would love and that I personally love. Thank you guys for watching this video, I hope you enjoyed it and I'll see you in my next video, bye.


Welcome to another live video here on Narc Survivor. In this live video, I'm going to talk about how you can recover quickly from narcissistic abuse. And when I say quickly, I don't mean it's going to happen overnight, but in many cases, people haven't made much progress, even after years have gone by. And I believe that you can make a lot of progress, even within just six months to one year. Naturally, when you're coming out of the relationship, you may go through a period of isolation. And there's nothing wrong with that. You may want to take some time to yourself. You may not feel like talking to people, but at some point, what's really going to speed up the healing process is when you get out of your comfort zone. As long as you stay comfortable, you're not going to make much progress. But I think the reason why many of us stay in our comfort zone after experiencing narcissistic abuse is because we're afraid of the consequences. We've been told to just stay in the box. Maybe they targeted our self-worth. They made us feel like we're not good enough, so we just keep it to ourselves. We don't try anything new. We don't go back to any of the things that we were passionate about before we were with the narcissist. When that's what's really going to heal you, your passion for life, just being at peace with yourself, being one with the world, with the collective consciousness, instead of continuing to exist in separation, which is what the narcissist wants you to do. So, just going out for a walk, whether it's a forest or coming out by a lake, this can be very healing for you, somewhere where it's peaceful, somewhere where you don't have to think.  They keep you thinking all the time, and that only prolongs your healing. And while it's understandable in the beginning, you may be thinking a lot, you may be analysing what you went through, but as time goes by, you want to get out of your head.   When what's really going to heal you from this is when you get out of that and you start finding things that you are passionate about, things that really mean something to you. And that could be anything, really. It's different for different people. I can't tell you what to do and what's really going to push you past this, but I can help you to find out what that is. For some people, it might be playing a musical instrument. It might be playing the guitar or the piano. It might be yoga or some other form of exercise. Or it could be a sport. It could be cooking. It could be making YouTube videos like I do. I think what it is most of all is it's something you're passionate about. It's something that gives you the opportunity to express yourself. Because while you're with these narcissists, you never get that opportunity to just be yourself, where you can express yourself, where you can just be you, where you can just flow freely through life without the fear of being judged, without the fear of being criticised, because that's really what shuts us down and locks us into this cage where we just feel helpless. We just don't know what to do. Because if you can't be yourself, what are you supposed to do? And I think that's what really drains the life out of us. It just crushes any opportunity of happiness is when we can't even be ourselves. Because you can't be yourself around a narcissist.    Because naturally if you're dealing with a narcissist, the odds are that they are going to be in opposition to any sense of your individuality or free thinking. Because they want to call the shots. They want to make the decisions. They want to dictate to you how to think. You can't think for yourself. You can't make your own decisions.  You feel like maybe the narcissist was right. Maybe I am no good. Maybe I do need someone else to make decisions for me. Maybe I'm not capable of anything. And it just sets you back. And that's why I say to really speed up the process on your healing journey is to just find something that you're passionate about. Find something that means something to you. Find something that gives you the opportunity to express yourself. Because while you've been around the narcissist, you haven't had the opportunity to do that. You can't coexist with a narcissist without being under their control. They can't have you thinking for yourself, making your own decisions because they look at it like that's time to get away from them. They need to occupy all of your time so that they can milk as much out of you as they can. But once you've moved on from the narcissist, this is what you can do. This is how you can speed up the process of healing. And I'm not saying it's the only thing that you have to do. Of course, you should be speaking to a professional, someone who understands narcissistic abuse, someone who can move you through the stages, through the denial, the anger, and more into acceptance. Because that's what's really going to heal you. So it's good to have a coach, whether it's me or someone else. But if you would like to book a session with me, you can go to NarcseFiver.co.uk to do that. But of course, I would also recommend speaking to a licensed professional, someone who can help you to heal the trauma. And for that, I recommend a service that I have used which is better help. And you can access BetterHelp by using my link which will also help to support our community which is betterhelp.com slash NarcseFiver. And of course, BetterHelp is not only very effective, which I know from using it myself, but it's also the most affordable service that you will probably find. You can have four one-hour sessions and unlimited messaging with a therapist or doctor for just £140 a month. And it's great, you know, just have someone there who understands someone who can help you to heal from the trauma.


Hi, I'm Amanda Jordan at Rickety Roo, and today I'm going to be talking about how to create kick-ass local landing pages. I talked about this topic at Mosconn in 2022. I did some research on the top 50 pages in the U.S. and home services businesses that ranked on the first page. And then I reviewed those pages and reviewed and analyzed the features that they were using on those pages to determine what makes a winning location landing page. So the most popular features are listed here, 61% of the home services businesses that ranked on the first page had reviews on their location landing pages. I think that's pretty much a given now, even though only 61% of there is 39% who were not doing it, but that's pretty much a given in my opinion that you should have reviews on that page for any other sake other than being able to use the structured data for it for the conversion rate optimization, just providing users with a reason to trust you and believe that you're good at your job. So just for any other reason, that would be the basic reasons why you want to do that. 32% had a unique value proposition, so something to set them apart from their competitors. I think that's actually very low, and that should be something that really all businesses should be striving for is setting themselves apart. You don't want to just be another site that's saying, hey, you can give me money for my services. You wouldn't want them to know why they want to choose you. And that is something that you do in your unique value proposition. Only a quarter of them included any type of sale or coupon, which is a huge miss opportunity. So if you're seeing that your competitors aren't offering coupon or sale, that's an opportunity for you to snag business from them after shopping around and going to multiple websites. If you're the only one that is offering a coupon, they don't know that you might be, I don't know, 10% more expensive from your competitor until they call and get a quote. So if you're offering 15% off, you may actually snag a customer and only have to get them 5% off because they don't know what your prices are compared to your competitor. So that is a good way to get more clients in, get more conversions. And then only 18% had awards or recognitions mentioned on the page. I think that's a miss opportunity as well because those are trust signals. Those show people that they can trust your business, that you're recognizing your industry, that you do good work. So these are the features that I found on those businesses in the top 50 most populated cities in the United States, where they ranked on the first page. These are the features they were using the most. There were some standouts where they had almost everything you could think of as far as features. And then there were others where they were getting lucky, like a little competition at their business that had been for a really long time, around for a really long time. You didn't have to put in that much effort to rank well because everyone knew them and they're almost like a fixture in their community. So if you put in that type, look for plumbing in that city, that's just what's going to show up. So really when you're thinking about your location pages, you should be trying to answer these questions. I put them here because I think like I detect them a lot of times when I review sites and I like true crime a lot. So I think about it as kind of putting together, what am I looking for? Almost, almost like clue. Like you want to know what's going on, who did, what, where, when, why, how. So when you look at your competitors, answer these questions and when you're thinking about your own location landing pages, answer these questions too. Who are you trying to reach out to? Who are, who is your business, who are you? What's your business? What are you trying to offer them? What are their concerns? Why should they choose you for competitors? When are you the best choice for them? Where can they contact you and how can they contact you? All of these questions should be answered on every location landing page. If you're not answering all of these questions, then people are going to have to search around on your website to find those answers or they're just going to leave and go to a competitor who can more easily answer those questions. And if you think about it, this is a lot of what Google tries to answer in your Google business profile as well. So if you want, you should be matching up, not necessarily word for word what's on it, but think about the features that Google is trying to show in their own tool and make sure that those features also exist on your location landing page. So you want to answer these questions and how you want to answer them. You want to use these, these types of data. You want to use first party data. So you know your customers, you've worked with them, you likely have a CRM that you use. You want to use that CRM to accumulate valuable data and use it on your pages. If you know that certain times of the year, certain issues exist for certain amount of houses and you're in home services, say, you know, the summer months, you already know in the summer months, AC units are going to need maintenance repair. Put the percentage of homes that you serve with those issues during those months on that page in that area that's automatically adding unique content that no one else has. And that Google is going to see too as unique content that's data driven content that's interesting content. So not only is it something that is unique to your page and not more generic than content that people are used to seeing, but it's also something that may be interesting enough that people will share with others that may be used for other reasons as a source for other things as well. Third party data. So you want to use statistics, FAQ, things that you can find around the Internet that you know is true, that it's relevant to your business and it's relevant to that specific location so that you can be as unique as possible within your content without being duplicative at all. And then user-generated content. Your users should be leaving your reviews. If they're not leaving your reviews, you should be asking them to leave reviews. So that's one of the things that you can do. Comment sections on websites. There's lots of clients that I've had, especially in e-commerce where they have e-commerce and local together, where they have a comment section where people can talk about their experiences. Not like in a review place, would ask questions, give feedback on some things, things like that, communicate with each other, almost like a forum sometimes too. And that's a ton of user-generated content that's right there that you didn't have to write that's about your products and services in your business. And it will show up in Google as well, so it's more usable content that you could have someone else produce for you. And here are my do's and don'ts of creating a location landing page. I'm going to start with the don'ts because I see these issues frequently. Even when businesses have the best intention, when they've hired an SEO, when they've hired a marketing director, I see that they run into these issues a lot and they're hard to overcome. Not to be a little like, like it's, they are difficult. Duplicate content is a huge issue for location landing pages. Then engineered content are huge issues. But if you look at your data sources that you could actually pull from easily with scalability, so you don't have to worry about someone having to go through and write about unclogging a toilet 200 different ways, you can pull your data about how many house calls you've gotten from about unclogging toilets seasonally by area, by zip code, use that data on that page instead. And that's so much more interesting than saying like, hey, if you have a toilet, come call us. Like everyone knows that you want them to do that. You can make that page more interesting. You can have reviews about people who called you for specific types of issues on that page as well to facilitate that uniqueness and personalization for the user. So really the duplicate content and the thin generic content issues are solved if you use first party, third party user generated content. They will solve those issues for you. And it doesn't have to be something extremely labor intensive. A lot of this data can be pulled from Google Sheets and to your website. Don't treat it like a blog. That is another issue I see very commonly is that a business will say, we need a ton of content to get people to come to our location landing page. We want this page to rank really well. But they forget that it's also about bringing someone in and making them want to make a decision to work with you on that one page. It's the landing page. So they shouldn't have to go anywhere else to determine if they want to work with you or not. So by answering these questions, you give them all the information they need to decide that they want to work with you. And you want to do it in a way that's engaging and interesting. So you don't want to have a huge block of text with nothing breaking it up. You want to include those any sales or coupons awards, reviews, unique value propositions throughout that content to break it up, and give them multiple opportunities to decide that they've read enough, they've heard enough, and they're ready to work with you. So these are my steps and recommendations for creating a kick-ass local landing page. If you want to discuss more, you can find me on Twitter at Amanda T. Jordan.


Hey everybody yes we're back and it's Bob Lorenz, Jack Curry and John Flaherty coming at you we've got that great news coming out Tuesday night major league baseball has a plan to get back on the field in late July and we'll dig into it guys in depth but flash from a players perspective you heard the news what jumped out at you. Well I think the players right away the excitement level just goes through the roof guys because you look forward to having a season and to be quite honest with you all of these guys have been working out but they're not working out with that same intensity that they know they're going back to spring training next week so I imagine guys got up this morning got their workouts in probably a little more intense than they happen and you're just trying to figure out a way am I in New York this is gonna be easy if I'm not in New York how am I gonna get to New York but you're looking forward to all of that but at the same time you're a little cautious on what is this new reality going to look like when we show up at Yankee Stadium and we begin spring training part two. Yeah I think we're looking at an unprecedented unusual season that's going to be in front of us we all know the health and safety of the players and everyone around the game is paramount the coronavirus is going to hover over it but when you dig into the fact that baseball is back John I agree with you it's a relief it's a reason to rejoice it's a reason to celebrate cautiously and I think the team that evolves adapts and adjusts is the team that can win this season because every hour of every day there's going to be something you experience that you've never experienced before in a baseball season and you can't let those whatever you want to call them obstacles and pediments distractions you can't let that get in the way of you have to win a baseball game that day however you can get that yeah flash I think that's what stands out to me is the difference for players right it's a different level of discipline now as Jack mentioned extraordinary unexpected circumstances hopefully won't see anything like it in any season ever again but if you're a player you've got to find a way to adapt all these things stay disciplined when where you're staying where you're eating if you're on the road what level of commitment from an athlete is that going to take compared to what they give right now well I think Bob all athletes and players have this discipline about them that they know what their schedule looks like every day when you get into the rhythm of a big league season so the health and protocol issues are going to be part of that rhythm every day for these guys but I think they're going into it knowing there are going to be some positive tests along the way and they're going to have to jack's point these organizations these players are going to have to adapt and figure out a way to win that game on that night I just think that the pressure is on these big league managers because how do you manage now you can't manage with that long season mentality and I might not throw my best guy out there tonight because we have to save him down the road at the same time you want to keep players healthy on the field it's going to be an incredible balance for these managers and front offices to juggle yeah Jack we've talked about that already the fact that it's always been a marathon not a sprint for a season it's a sprint now it's 60 games and and to me that's part of the excitement of this season again we're not going to see anything like this ever again so that's part of the the new reality and a wrinkle that I think is going to be interesting we're almost every game in a way is a playoff game oh there's a ton of urgency to everything that's going to be going around every game that we see and to John's point I think a few things are going to happen first of all the teams with the best bullpens you're going to lean on your bullpen heavy this isn't an April situation where as John said well let's let's save Chapman and not use him today so I think it's going to be the team with a stacked bullpen I think roster depth is going to be vital I think having a killer instinct is going to be vital you're playing games in July and August you better treat those games as if you're playing in October and then if I was Aaron Boone on my wish list I'd wish I had three or four Chad greens going back to that bullpen point you want a guy who multiple times a week can give you multiple innings so I look from a Yankee perspective somebody like Eliza got and how important he can become someone like Luis Sessa even a Davey Garcia if he works himself into the mix those extra arms are going to be so vital right so flash let me ask you this Jack mentioned some of the players who could have a big impact or maybe in a 162 game season they wouldn't about the fact that for the Yankees they were scheduled to start the season without some players but given the Corona virus guys have rested up John Carlos Stanton Aaron Hicks James Paxton should all be back when the season begins in late July how much of an advantage does that give the Yankees I think it's a huge advantage from where they would have been a couple of months ago but you know is Aaron judge going to be a healthy player when this opening day comes that's still a big question mark and guys I just keep going back to you know in my mind what if a guy like Stan gets hurt and that's you know a three or four week deal I mean that's half of a big league season now so it's such a big situation but Jack I was thinking about when you were mentioning all of these things you know from a player standpoint these guys have to get off to a good start right away right I mean they it's not like okay I got a couple of weeks or a couple of months I'm a second half player I'll find my stride you have to get off to a hot start right away and if you don't the organization has to take a look and say we can't have the patience we might have to make a move here that we didn't think we were gonna have to make because we only have 30 games left in the season so when we're gonna be doing our shows I mean it's gonna be so many things to break down and analyze it's gonna be it's gonna be fantastic it's gonna be different and I know the traditionalists out there say oh it's not gonna be a regular season I get all that I just think 60 games is fascinating and getting to the playoffs and how you get there is really gonna be wonderful to watch we always knew 2020 was going to be different well once the pandemic occurred we all knew that so I say embrace what is in front of us a 60 game season could have a ton of excitement I use the word urgency and I use the word evolve and John you spoke about it from a player perspective how about this if you're a team that starts out the season two and 12 you might have buried yourself you might be done and that's why those those first 14 16 20 games whatever you want to say that's why there is an urgency and how about this factoid Washington Nationals were 19 and 31 after 50 games last year and won a World Series we all know you're not starting 19 and 31 and winning a World Series this season you're not gonna make playoffs if you have that kind of long slow start really changes the mentality now one of the other things I wanted to bring up there are some different wrinkles that we're gonna see and among them and this is where I don't want traditionalists heads to explode keeping it under the microscope of a 2020 season only it extra innings to help preserve players health speed up the games a little bit a runner will start on second base so flash this is intriguing to me I'm actually embracing it I know it's getting away from the norm they did in the minor leagues but now on a national scale in the major leagues a lot of people are really gonna get their first taste of this viewers and seeing this I find that a little intriguing for this year yeah I don't love it but I'm with you Bob I'm going to embrace it and see what it looks like and again I go back to managers and strategies does the sacrifice pump come back into play which really hasn't been a big thing recently in our game the pinch runner the speed factor is obviously gonna be huge does Aaron Boone hold the Tyler Wade in a tight game knowing that he might have a pinch running situation at second base and to utilize his speed so there are so many things that go into it you know and I understand the health and safety of the players and being able to manage bullpens you can't have 16 in games I get all of that so if you understand that part of it you try to embrace it and see how each club is going to take advantage in that type of a situation in a normal season guys I'm like John I would have scoffed at this rule this season this is not the year to complain about everything if you happen to be on that maniacal crazy side of liking to see pitchers hit which I'm not on that side don't complain about the DH this year by the way it's long overdue let's have the DH and that extra runner we keep using the word embrace it and John brought up a great point the player who will start on base in extra innings is the player who made the final out from the previous innings well for the Yankees if that's Luke Voight and you've got a Tyler Wade stashed away in the dugout you're absolutely pinch running for him you're going for the kill right then and there that's exciting that's baseball that's what we've missed for the last few months don't be screaming at your TV that this feels like little league or American movie scream at your TV I'm so happy that I get to see this again alright so final questions for each of you I'm gonna start with you flash again some of the new rules no spitting no tobacco no sunflower seeds you are allowed gum would that be tougher for you or on an off day for you non-playing you're gonna be sitting in the stands socially distancing which would be tougher for you to kind of wrap your head around well I mean the the instinct right away for players you know whether you shoot tobacco gum or seeds is spitting whether it's disgusting or not it's all part of the game and it was just something that you didn't even think about when you're out on the field so I think that's gonna be interesting but the whole idea of playing games in stadiums where there are no fans is going to be fascinating for me because I think on TV it's going to look and feel a whole lot different but but I actually think that picture batter confrontation where where you're just locked in in the battle that's never going to go away so I still think there's gonna be intensity out on the field it just might look and feel different from what we see on TV and Jack as we talk about doing things differently in this season hopefully this season only gonna be a completely different perspective for reporters covering the game as well the way we do it as broadcasters and I wonder your thoughts isn't this a time for the broadcasters in the league to get creative in terms of how we cover these athletes ask them questions etc what are your thoughts on that yeah I think creativity is key I think being clever I think being intriguing being interesting I think knowing the players and having interviewed them in the past and knowing some of their habits and some of their routines I think that will help you going forward if you see an at bat from DJ LeMayhew and he inside outs a ball to right field and you had a conversation with him about that a year ago well that's the time to resurrect that conversation here's what he said about why he likes to do that at Yankee Stadium I keep using the word and break the ball but that I think that's for all of us we're going to have to cover the game differently a lot of the interviews are probably going to be just like this via zoom but I think the fact that we have baseball back that we have something to talk about and then we have some clarity in front of us I think that is a reason to rejoice and guys just about time to dust off the suits and ties let's just hope the slacks and the pants fit thanks for joining us we'll see you next time good to see you guys


President Bola Tinnubu today arrived at the Muritela Muhammad International Airport in Lagos, from London, United Kingdom. He arrived at exactly 5.30pm. It was received by supporters and high-ranking members of the All Progressives Congress, including the Lagos State Governor Babaji Desawulu, the Chief of Staff to the President Femi Bajabiamila, Acting Inspector General of Police Ulukaya Di Egbetokun National Security Advisor Nuhu Ribadu, Senators and Representatives from Lagos State. The President's head on June 22nd, 2023, made his first official trip overseas after his inauguration as Nigeria's new leader on May 29. A statement upon his arrival, the President thanked the Nigerians for the overwhelming reception accorded him in Lagos. Tinnubu is scheduled to join fellow Muslim faithfuls for the Eid prayers tomorrow at the Obali Day Eid prayer ground located at Dodden Barracks, the former seat of the Nigerian government.


There we go Because a lot of you and we all ready of course expected before we started the course a lot of you will use the genomics chromium method in order to generate single-cell tritotomics data Because so many people are using it we can go we can spend a little bit time to go a little bit more in depth It's actually going home when you generate the data and if you understand what's going on that also makes things easier to troubleshoot for example so it's about the Think about seven or eight flight or so just some visualization on on then it's you know mix in general Or actually specifically with chromium method So you have seen this MSB4 in a previous Presentation just to as a recap. So what do you have? What is the main invention? That allows you to generate single-cell tritotomic data with this chromium device So this room device is kind of a cell sort there where the cells and gel beads move through through the oil actually and The gel bead and the cell you hope that they are captured in a single DEM and Because you associate a single gel bead with a single cell you get and this is the the the gel bead has specific barcode for the bead you can actually Find out later on after sequence Sequencing which we actually came from which cell actually which we belong together in the same cell This image It looks like in this image that you actually a Lot of cells move through the device more than beads but actually in the case that Cells are moving way more sparsely through device than the beads and because you do that just like John You expect that if a bead gets associated with a cell then it's only one single cell just because very few cells move through the device so on the gel bead is that a sphere is completely packed with oligos and And those oligos they look very similar for all of them So they all have the same adapter sequence within a delby all have the same barcode forces difference between delby UMIs they differ between oligos more about your mice later on and Then we have the poly a tail or actually kept your sequence and nowadays We have more different capture sequences not only a poly a tail But also other sequences that allows you to capture for example These indexes related to antibodies Okay, so in this DEM what you get is The cell gets lies you capture Messenger RNA that is poly adenylated with this poly T oligo and That means that all of those capture transcripts from a single cell They have an identical Within the same cell and you need barcode because all the barcode oligos on a single delby are the same So when you have that Then you get a reverse transcription that still happens within the GM then the GM's are broken down and You get fragmentation step meaning that the messenger RNA get broken into smaller pieces Then you get the ligation of the primer. So the other chance basically of the the library is basically generated and You get an index PCR a measure what you end up with is this contract over here You have to be five and the P7 that are required for Illumina sequencing in order to be able to renew the sequencing lane we get the Read one and We do which are the typical Illumina adapters get the barcode that is specific for the jail beat the UMI the poly DP and Then the part that is actually sequence the part of the individual gene the other adapter You get a sample index. So that's a typical index that is sequence by Illumina in order to be able to be multiple samples and of course the P7 so When you start sequencing this contract what you get is of course the adapter sequence for the forward read and the adapter sequence of the revert read The depth of sequence word for it read is used to only sequence the barcode that is specific for the jail beat and UMI Anything else is kind of irrelevant because that there'll be only poly T or poly A and The other adapter is used to sequence the actual Sequence of the transcript and that is of course used later on to do the alignment for example and it also We also usually get a third Post Q12 or sample so we get rebound we do entry three or I one actually and that contains the sample indices So how does it look so you have brought your library or maybe your sample to the sequencing provider They did the Library prep and the sequencing for you usually you end up per sample or actually for sequencing lane you get three Post Q file we drawn entry to read wrong with only the Barcode and UMI we do with the actual transcript index and I won with the sample barcode If you use dual indexing you can sometimes or even get an I to Sometimes that I won and I to are both missing and then usually in these is they are added to the fostering type So we are actually added to the fostering files themselves the R1 and the R2 So after a sequence in so you have the role of a few files what's next so then what's next is of course We have to figure out with which read actually came from with cell or actually which we belong together in the same cell the alignment and then the quantification for gene so yeah, yeah at some point you want to end up with count per cell per gene and Also, then figure out what is a cell and what's not because they will also be background RNA in there more about it in a latest slide So for these four steps typically for Tenex data What you can do it use a software called cell ranger that is Provided by Tenex genomics and last of all those four steps So it's a single command relatively straightforward to use especially for model organisms and You just run that on your fostering files and what you end up with what you're mostly interested in usually for downstream analysis Is this countable with cells and and teams? Alternative to use in tar ranger are using star solo Actually, our solo is used by tar ranger under the hood let's say My apologies for the dog Thank you very much Starts getting active Somehow that's an important reason Another alternative is using elevin so that's from the people who also developed Selman and has a bit of a different way of Generating down tables, but I guess tar ranger is really the most used Software programs to actually perform all these four steps To come from read data to actually account data count table Of course, what cell range it needs is a reference sequence because it's one of the steps So step two is of course aligning the sequencing read to a reference genome So you need a reference sequence and it has to be relatively specific In a way you can just use your reference genome and and the DTF but the DTF has to be in a specific format Can download it from the 10x website for human and mouse. So that's like the the the standard Would be the standard reference to you So just down to make it and download it and you can get started for other Organisms you have to make a custom reference with cell ranger and gay rep Also relatively straightforward to run if you have highly standardized genome and highly standardized DTFs if you have any excellent marketing like DFP, so you have done experiments with the DFP in there and you want to actually Estimate the expression of DFP.  So You have to add it to your phosphate and enter DTF and then again run cell ranger and gay rep in order to create your reference And then you're good to go If you have other features other than than genes for example proteins You will need a feature barcode reference. You've actually specified which barcode is associated with which protein And then cell ranger can also work with that So then about UMIs. So what are UMIs and why do we use them? So UMI So the abbreviation UMI stand for unique molecular identifier and basically it identifies authority Kind of in the name it identifies each molecule uniquely meaning it identifies each of those constructs We are creating to do the actual sequencing uniquely Which means that Before you do the actual sequencing on those constructs we create so with the barcode and the actual sequence in there And you imagine everything in there we do a PCR because without PCR We do not have enough of those constructs in order to be able to do the actual sequencing so we have to do a PCR and Then everything all of the molecules or all the fragments construct after this better work I get all the construct originating from the Same initial construct will have the same UMI Meaning that if let's say you are going to sequence your entire library very deeply at some point You will just sequence the same construct over and over again with the same UMI They do not get any more information out of it so we want to What we basically want is to have sequences in there with unique UMIs because then we know We are drawing a part of a transcript that is not sequence before So therefore we have UMIs in there also because Usually for 10 economics as you remember well the Libraries you create are usually not very complex Which means that you very quickly are sequencing the same construct over and over again But counting these UMIs you're actually correct for that So you do not count the actual alignment as you may be used to for an AC Or actually count the number of individual unique UMIs per gene So you directly correct for sequencing the same construct over and over again So when you have run a cell ranger you get a report a very nice report. I always look at it quite extensively Which gives you the main statistics that are important for well the next steps in your analysis one of them of course is for example the number of cells you have that are called they are Considered by a cell ranger as actual cells You have the number of my mean number of reads for cells So that tells you about how many reads you have generated for a cell and the median number of genes for cells So how many genes you are actually measuring? Can of course differ for a tissue some tissues you can expect more Reads more genes to be expressed than others And some of course and it depends very much on the cells there are in there Then you get information about the total number of reads you have for that run the for example, how many Reads aligned to that genome can be important, of course or is usually very important and how many reads Have met to the excerpt so to external ingredients to the actual teams for human amounts usually the The genome is well annotated or a very well annotated which means that Many of the reads are expected to align to an exonic region to a gene If you work with non-modal organisms usually annotations are not so so good meaning that Reasons that are actually genes are not annotated as being a gene maybe a UTRs are missing for example Typical problem is that pre-prime UTRs are missing which means that actually the the position That you are aligning your reach to is just missing in your reference genome And then you get very low number of reads that maps to an exonic region and therefore you do not Take them into account than estimating mean expression. So these numbers over here about the mapping can be Or are very relevant and especially if you are working with normal organisms are very important to check You get some information about the sample about the chemistry that you that's usually often detected by by cell ranger That's very convenient And of course about the cell calling And about this You might have noticed I own a dog and sometimes a bark my policy. So about dog calling Very important part of running cell ranger So what happened is let's say you have your droplet and in your droplet in ideal case most of the case You both have a cell and a beat However Usually you also have free messenger RNA. You have free a messenger RNA in tissue in general And if you have been this this associated Dissociating your tissue very often you break down cell you do cells get broken And they release messenger RNA and if that occurs very frequently What happens is that you get a lot of background messenger RNA in there and that can be a bad thing because you do not know where this messenger RNA Where it comes from? so very often it also happens that you capture only background RNA to get in in your In the droplet meaning That you have well your measuring messenger RNA, but they do not come from the same cell so well What cell range to try to do is it compares or it looked at the distribution of the number of you my first cell and it orders that in a crop and Then looks for a steep drop because what you expect is that Droplets with a cell they contain a lot of messenger RNA and droplets without the cell They contain very little messenger RNA. However, if things didn't went very smoothly with dissociation, for example You expect a lot of background messenger RNA and then it becomes very difficult to separate Droplets with a cell and without a cell because you have that much background RNA in there and then you get plots like the The bottom right where you do not have a clear drop in The number of you my first cell and then it becomes very difficult to figure out what the cell and what not what has only background in there Typically to solve this or you just have to deal with it as a biophonetician So often to me if I showed it to a customer, then they say yeah Can you do anything with it? And I say yeah, well you can just try to get cells out of there Have a try multiple threshold for example and see whether you get Biological meaningful analysis out of there, but still it becomes very difficult to know what is a cell and what's not maybe in a UMAP later on You can find signatures It's not too breezy to work So some other parameters to look at in the cell range report is of course the number of captured cells And of course usually you won't quite a bit of quite a few cells because the more cells you have The the better that this offer for example statistic the more likely it is that also rare cells end up in your sample But you can often not have too many because if you have too many cells then the chances are much higher that you have Single be associated with multiple cells And of course if you have single be associated with multiple cells, then it's not single cell transcriptomics anymore than it to cell so There is a relation between a number of cells and the number of expected doublets So if you go up to 10,000 tell you expect about seven percent of those Beats being associated with more than one cell And of course you do not want too many of those because they are actually quite difficult to identify the doublets So in principle you could filter for it there itself there for it, but it's not super trivial to do So you want to be in between these thousand and eight out and so not two little cells because otherwise well Of course, you do single cell Transparency can use 10x have quite a few cells in there, but also not too many because otherwise you get doublets Number of reads per cell depends very much on the library complexity you have meaning that At some point just generating more reads per cell does not give you more information because you're sequencing the same construct all over again over and over again But you won't end up in order to be able to Sequence the entire trust with them as much as you can sequence from that cell Typically it's between 30,000 and 100,000 weeks per cell And then sequencing saturation tells you about whether it could make sense to generate for example more reads So the sequence in saturation if it's low, which means that you do not sequence the same construct multiple times, which means that it can make them to Generate more reads if the sequencing saturation is very high That means that you have been sequencing the same construct over and over again and then also generating more reads doesn't make a lot of sense And of course, I already spoke about it the number of weeks mapping through the genome and through the transfer And typically what you can do for example is change your transfer If you're working with a non-multi-organism look at the annotation of for example the three primary pairs If that's not a very good annotation they might want to Change it by for example extending the gene length


Hi everyone, Joe Projasky's casewrakes.com here. A little lozzage in my mouth, sorry if I'm a little mumbly. And I'm coming out to the brand new release, 2021 Panini Chronicles Football Six Box Half Case Picker Team 2. Second half of the case that we're going to do here, remember I did the first half not too long ago, put those little Wolverine slash marks there so you know they're from the same case. So for Picker Team 2 on Wednesday the 13th, big thanks to this group here, thanks for getting into it. And let's see what we got, first half was pretty solid, hopefully the second half continues that trend. Good luck everybody. Just in the interest of time, cards like those quarterbacks right there, we're just going to sleeve and our team will be able to top load those before they're sorted and shipped out to you. Alright, thanks everybody, here we go. Nice Trey Lance and Cadarius Tony, Relic, 132 out of 199. Zach Wilson red to 99. Grant with the Jets, nice Trevor Lawrence, Jesse last spot mojo with that Trevor Lawrence. He had a nice first half, let's see if that continues in the second half. Trey Lance, Niners, Jay Gragg, Zach Wilson, Jets, Grant, and that Cadarius Tony is numbered 78 out of 99. And we've got Jesse Sappalo, 4 out of 10, totally certified signatures. That is for Jay Gragg's and the San Francisco 49ers. Nice Justin Fields for Rick T, won that team in the filler, thanks to Legacy, Mac Jones, and a Davis Mills to 199 for the Texans, that would be for Ron. And as Zach Wilson, is this an autograph? Yes, in the Legacy design, rookie auto for the Jets, that's going to go to Grant, won a spot in the team random from a filler, and then won the Jets. So that's a nice little parlay there, Grant, 58 out of 99. Hopefully a good season for him. I'm rooting for him, it seems like he's in the best shape of his life. The classic, I'm in the best shape of my life. By all indications, before you think back to the draft, everyone said, or most people said that the Jets had a great draft, if not maybe the best draft of all the teams. I think they had a pretty solid off-season too, you know, the coach, Robert, I almost said Mohamed Salah, that would be the forward for my club Liverpool. No, the Robert Salah, you know, he's got a, he's not a rookie head coach anymore, he's got that crucial first year under their belts, under his belt, him and his staff, hopefully onwards and upwards. Right, exactly, right, because it's like bands are like, hey, this is the best album we've made. This is like, when you know it's like not. This year would be shocking, old school, 67 out of 99, I don't know how many late era U2 albums were like that, one of the best albums of our career. New York football giants, Aaron, other Justin Fields, Matt Jones, Zach Wilson, and T-Law, Trask, and Kyle Trask, Triple Relic and Autograph, 46 out of 75, Momentum, Triple Relic and Auto, maybe future Tom Brady right here, this is going to go to Angel Acevedo, who got the Buccaneers in that random. Zach Wilson, Chuba Hubbard to 399, and we got a Joe Burrow Relic, 69 out of 199, and Jeremy with the Bengals, Matt Jones, Luminance, Kyle Trask, Orange to 75, Justin Fields, score, Jalen Waddell to 49, to 99, Micah Parsons, Odufe Oesh, Jalen Waddell for the fish, the Dolphins, they're not fish, they're mammals, that goes to Dean, one of that spot, Justin Fields for the Bears, that's for Rick, Kyle Trask, Anhel, Buccaneers, Patriots, Han has all these Matt Joneses, a lot of Matt Joneses going your way, Han. Patrick and the Panthers, Grant with the Zach Wilson, all the Zach Wilson's, here's the numbered Kyle Trask right here at 99, DC saying you need a Parsons Prism Black Auto, I do love that Prism Black design, I'll try to find one of those, two boxes in, here's the third box, almost half way through this half case break, alright we got a Devonta Smith Relic, 10 out of 399, exciting Eagles playmaker going to Travis and the Eagles, Davis Mills for Houston, Ron, Trevor Lawrence, Legacy Red, 60 out of 99, Mac Jones, Antonio Gibson to 399, and a William Perry autograph, 25 out of 100, big man there, old school auto going to Rick and the Bears, that sticker can't contain William Perry, more Mac Jones, Tray Lance, Jake Rags with the Niners, Ian Book to 199, Kyle and the Saints, and Kellen Mann, 1 out of 25, Vikings, that'll be for K-Shine, Safford to 99, Tray Lance, Penini Black, all right we are half way through this half case break, good luck, we've got a Dave Krieg, another old school totally certified auto, 6 out of 50, Seahawks that's going to be for Adam, kind of think they should bring those old colors back for a season or two, Tray Lance, score, J-Grags, there's some Micah Parsons right here, we're looking for some autographs, out of 199, that'll be for Francisco and Dallas, Jamar Chase, 39 out of 99, nice, a lot of times he's a redemption, here's a nice little nice sticker auto here, he's got some good penmanship, Bangles, Jeremy H, picking up the Bangles straight up, gets the offensive rookie of the year, T-Law, Rashad Bateman to 199, he might get no more Marquis Brown on the Ravens, he might get some more looks Rashad Bateman, he was pretty highly touted coming out of college but like much of the Ravens last year, a lot of injury issues, that goes to Simon in the Ravens, Tray Lance, Flux, Trevor Lawrence, Clear Visions will be weird not saying Big Ben on a weekly basis, Zach Wilson, Timeless Treasures, Relic, 191 out of 399, Grant with the Jets, Justin Fields, Davis Mills, nice Trevor Lawrence, Panini Black, Parallel, Zach Wilson to 75, this teammate Michael Carter, love this design right here, Jesse Garcia with the Jaguars, wish we could see like a one of one or a train whistle hit of that, that'd be awesome, JJ Watt to 399, Justin Fields in that legacy design and Jaylen Waddle with Tyree Hill on the other side of the ball that he could have a big season too, already had a nice season last year, alright two boxes to go, good luck, Justin Jefferson to 399, and Kine in Wangwu is your autograph, 44 out of 99, Great Iron Kings auto, that is for Keshon and the Vikings, out of 199, Teres Marshall Jr., and Travis Etienne Jr., Triple Relic and Autograph to 25, Runefern hopefully he's 100% healthy, can contribute to the team, that'd be good for the hobby, that'd be good for Jesse, and the Jaguars, last bot Mojo, 70% of the time hits 100% of the time, Saquon to 49, Michael Carter to 199, Grant with a piece of his jersey, Trevor Lawrence, Trevor Lawrence, Tray Lance Purple to 199, and Final Box coming up, Tray Lance is pretty cool Jay Greggs, I like that, really pops there even though it's just out of 199, alright Final Box, fingers crossed everybody, good luck, Fields and Daniel Jones to 399, and Teres Marshall Jr., Autograph for Carolina, Patrick and the Panthers, it's 11 out of 99, Patrick, Zach Wilson, Davis Mills, Kyle Tras to 199, Zach Wilson, Travis Etienne Jr., Jersey and Autograph, another one, 37 out of 75, there you go Jesse and the Jags, last bot Mojo, Franks for the Falcons, that's for Josh, Clyde Edwards DeLair, piece of his 199, Aaron and the Chiefs, Justin Fields, and an Eli Mitchell Gold Vinyl, 3 out of 5, I was saying it would be cool to see a low numbered parallel for those prison blacks, and here it is, that's awesome, that looks really sharp, had him on my fantasy football team last year, really solid season for Jay Greggs out of 5's and under train whistle, all aboard the Big Head Express, whoo-hoo, it's a view, and a Zach Wilson legacy, and there you go ladies and gentlemen, that's it, that's 2021, Chronicles football, Six Box, pick your team too, quick little Rurikap, nice Jamar Chase, live Jamar Chase, William Perry, Zach Wilson Autograph was cool, that legacy auto, and of course the Gold Vinyl Eli Mitchell, and plus plenty of other, bunch of other cards that we've sleeved that are going to be top loaded and all that stuff, thanks for watching, thanks for breaking with us folks, I'm Joe, I'll see you next time for the next break, bye bye, jaspyscasebreaks.com


Well, hello and welcome to Jonathan from the heart. I'm Jonathan Asley from JonathanAsley.com. And I'm so excited to be shooting this short video for you today. It's my first video for 2021 and I'm really excited to be sharing this with you. So I want to celebrate the new year and also say, let's hope, now that we put 2020 behind us, let's hope 2021 is our time to meet the love of our life. And that's our topic today. Four days to meet the love of your life. Or is it gonna be like this? Four days to meet the love of your life. And this really works, okay? So really quickly, if you're new to my YouTube channel, hit the subscribe button, hit the bell for new content. Also, if you like this video, these are exactly the video, this is almost exactly like the videos I shoot in my private group called Midlife Love Mastery. Check out the link below where I answer the questions you ask by shooting videos three days a week in our private group, Midlife Love Mastery. All right, happy new year. Let's get started. Four days to meet the love of your life. All right, I'm gonna take this off now. So I wanna share with you personally, I am a recovering codependent. And I would say on the scale of love attachment style, I'm an anxious attachment. In other words, I become very needy. I have this subconscious belief that I need someone else to love me to feel good about myself. I've operated from that perspective for a significant part of my life. And I said recovering codependent because I've been working deeply on shifting that. I've been working deeply on shifting out of that narrative for myself. And this plays such an important role when it comes to meeting the love of your life. So let's talk about what the love of your life is right now because I'm gonna teach you how to meet that person in four days, okay?  The love of your life, bear with me one second. And I hope this works. If you look in the camera, okay, that's a mirror. Okay, this is a mirror. This is a mirror, okay?  I want you to find right now or at the end of this video, I want you to go to a mirror.   And because that's what it took for me to get into this place. Now, many of you might already feel this way, which is great. Many of you don't feel this way. Some of you might feel this way from an entitled place. And really that's just our ego trying to rationalize things. But if you're seeking genuine, true companionship, connection and intimacy with another human being, then I encourage you to find it within yourself first. And I'm gonna teach you how to make that happen in four days, okay? In four days, all right? And this piggyback's from some of my other videos I shot. So get excited about the next video that's coming because it's gonna help you manifest that great person in your life. Okay, four days. First off, let's establish that a day is 24 hours. So one day is 24 hours, okay? So four days is 96 hours. So I'm gonna ask you to invest the next 96 hours of your life. But I'm gonna make it simple. You only have to do one hour at a time, okay? One hour at a time. So really it's 96 days. But 96 hours is four days, okay? And you can do that. People say they spend something like 1,000 hours in traffic each year and 500 hours standing in line each year, although with the pandemic none of that was happening. But we spend so much time wasted. How about you invest one hour for yourself a day? I like the way Tony Robbins calls it the hour of power. He's all about power, the hour of power. So what are you gonna wanna do with this hour? Well, I wanna share with you what got me to the place of where I'm at today. And ladies, I can tell you, I'm no longer dependent on someone else for my happiness. Gosh, that feels so good not to feel dependent on someone else for my happiness.  Am I right? If I am, please post a comment below. So what we're really gonna be talking about is how to join the self-love club, the self-love club. I've got my mug today that says love yourself.  Anyways, okay, how to join the self-love club.  What the Heck is Self-Love Any? This book was birthed after the loss of my 19-year-old son, Connor, in 2018. I'm coming up on three years and that's gonna be tough. It is just getting tougher each day to navigate life without him. And yet I'm also finding that depth inside of me to be able to lean into, lean into love, lean into self-love. Now, why I want you to get my book is it takes you on the journey of how I got to where I'm at today. It's self-love is a vaccination to emotional chaos. So check out the link below to self-love the book to get my book, okay? Now, this next book you have to purchase. This is like my, I don't like the terminology, but this is my Bible. It's called The Untethered Soul by Michael Singer. The Untethered Soul by Michael Singer. There are 19 chapters in this book. So part of your instruction every day is to read only one chapter a day. One chapter, that's it. Take you 15 minutes. Then I want you to invest about another five minutes right afterwards and just meditate on what you read. Or okay, meditate on that. Now, this is gonna feel like spaghetti. For the first four or five chapters, it's gonna be hard. You may have to reread a chapter over and over. You might read a page and reread it. That's okay. That's really okay. I promise you get through this book. In fact, read it twice. Read it often. Put it on your coffee table. I gave this to a dear friend of mine who is a skeptic on the whole spirituality, personal development, self-help. After reading this book, he bought five copies to give to his children, to his friends. This made a difference in another person's life and my hope is it makes a difference in your life. So again, one chapter a day, okay? One chapter a day and a few minutes of meditating. That's only 15, 20 minutes. All right, so you got another 40 minutes. I want you, you don't have to do this all at the same time, but I, and by the way, you should be reading my book along the way. I definitely want you to get the Hoffman process, the Hoffman process book. This is similar to the experience I went to the actual retreat, but this is just right off the bat. You're gonna be reading this and going, oh my God, I got so much stuff that needs healing. I've got so many childhood wounds and traumas that need healing. And when we heal, we begin to love ourselves. That's what this is all about. And it doesn't mean you're fully healed, okay? It's not like you're fully, I love myself 100%. Just begin a practice of loving on yourself. And so invest another 15 or 20 minutes in this book. I highly recommend it. Okay, now there's another book. As you're doing all this work, you're investing an hour a day. I want you to then get this book. I never talked about this before. Creative Visualization Workbook by Shakti Gwani. And I'm butchering the name, so please forgive me. This is a workbook, okay? This is a workbook. Look it, you can spend thousands and thousands of dollars with coaches and you can do a lot of this work for 15 bucks by buying a couple of these books, or 50 bucks for buying these books. Now, if you need help on how to vet men, that's my area of expertise. You will have to hire me. Check out a link to a free discovery call because that's my area of expertise is helping you figure out men. But I want you to figure out yourself first because then your intuition will be highly tuned. After these 96 hours, your intuition is gonna start working at a whole another level.  You're operating from a place of self-love. And let me tell you, whether you find a romantic partner in your life or not, whether you find it or not, when you find yourself and when you're loving on yourself, it's not a desperate need. And the reality is, and I'm gonna give you some real reality because I know a lot of coaches will sell you on the idea. Hey, as we get older, this isn't easy. This isn't easy to find a life partner. I don't like the word easy, but statistically, it might not happen. This is why the best relationship you can have is with yourself because the reality is, is the relationships we have in our lives determine the quality of our lives. That's what Esther Perrell says and I love that. So when we start with the relationship with ourselves, we're already setting ourselves up for a great quality of life. And just remember, self-love is the antidote to inner suffering. Self-love is the vehicle to navigate life through inner peace. And ultimately, that's what matters most. It doesn't matter. You're gonna come back and the earth, you get to do this over and over and over again from a spiritual perspective. You get to practice this. So I'm suggesting do this practice for the next 96 hours. Let me know if, hey, if you're willing to do this, please post a comment below. I want you to track it for me. I want you to come back 100 days later and let me know how you're doing 96 days later and let me know how you're doing. Are you willing to do this? I hope you are. I want you to, because here's the thing, I can give you a lot of advice about men and why they pull away and you can listen to the whole feminine energy crap, which is really just trying to turn you into a Southern bell, you know?  And so you've attracted the guy in, but those only last a few months.  That's the best part. Is this sinking in? Please let me know. Post a comment below. I wanna hear your thoughts on this. Are you willing to join the Self-Love Club? By the way, if you wanna get this t-shirt, go to Amazon.com. There's a link below to my books and it'll take you to Amazon. But there's a just type in Self-Love Club t-shirt and you can get this as well. All right, I hope you found value in this video. I hope, I'd like to hear your thoughts on it. Please post a comment below. As always, I'm gonna wrap up my videos as I wrap up this video as I always do.  Self-love. I'm gonna reach into the camera and give you a hug of love. Oh, listen, I wanna go do something really quick. Tang tight. Also, to celebrate the new year one more time. I want you to do me a favor. Reach out to a friend.  Reach out to a pet, a pillow, or a teddy bear and give it a big, gigantic hug for 2021 because let's face it, hugs are a great source of love and we can all use more love in our lives. I wanna thank you so much and wishing you a super duper wonderful day. Bye-bye now.