Chromatin Immunoprecipitation of Histone Variants

35 OD₆₀₀ U of log phase wild-type and H2A-E57A mutant cells were crosslinked and sonicated in biological duplicate using the protocol described in (76 ). Histones were immunoprecipated from 1 μg chromatin and 1 μl of anti-H3 (Abcam, 1791) conjugated to 20 μl protein G magnetic beads (Invitrogen, 10004D) per reaction. Libraries were generated using the Ovation Ultralow v2 kit (NuGEN, 0344) and subjected to 50-bp single-end sequencing on an Illumina HiSeq 2500 at the Fred Hutchinson Cancer Research Center genomics facility. We used bowtie2 to align raw reads to the sacCer3 reference genome (70 (link)). Reads were then filtered using SAMtools (71 (link)). Bigwig files of input normalized ChIP-seq data were generated from the filtered bam files using deepTools2 (73 (link)) and dividing the IP data by the input data. Matrices for metaplots were generated in deepTools2 using the annotation file from (77 ). The metaplot in the final figure (Figure 5G) is an average of two biological replicates after assessing correlation between the two replicates. To do this, fastq files of raw reads were merged and the above pipeline was repeated on the merged fastq files.