Transposon insertion sequencing (TIS) is a highly effective method used with bacteria to identify genes important for growth in any condition of interest. Previously, we adapted this method to identify essential genes of the yeast  Schizosaccharomyces pombe . Here, we describe modifications used to identify genes necessary for the formation of centromeric heterochromatin. For complete details on the use and execution of this protocol, please refer to Lee et al. (2020).