Chromatin Immunoprecipitation Sequencing Protocol

The ULI-N-ChIP-seq protocol^{65 (link)} was used to generate ChIP-seq libraries, with minor modifications. Briefly, chromatin was fragmented with 3.33 units of Micrococcal Nuclease (NEB), diluted in native ChIP buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA; 150 mM NaCl, and 0.1% Triton X-100) containing 1 mM PMSF and EDTA-free protease inhibitor cocktail (Roche) and sonicated three times with 5-second cycles on low power (Diagenode Bioruptor). Chromatin fragments were then split into two aliquots (~1000 cells/ChIP) and incubated with each of the histone methylation antibodies at 4 °C overnight. Antibodies used for ChIP experiments were H3K9me3 (39161, lot 13509002, Active Motif, 125 ng) and H2AK119ub1 (8240, lot 6, Cell Signaling, 500 ng). Following purification of ChIPed DNA by AMPure XP beads, libraries were constructed, pooled and sequenced as described above. Libraries were sequenced either on the NextSeq 500 platform with 76 paired-end reads, high-output mode (as for RNA-seq) or on the NextSeq 2000 platform with 66 paired-end reads, P2-output mode. ChIP-seq reads were aligned to the mouse genome (mm10) using bwa^{103 (link)}. PCR duplicates were removed by Picard-tools (http://broadinstitute.github.io/picard) and reads with low mapping quality (MAPQ < 5) were excluded by Samtools (http://www.htslib.org/). Processed reads were then used for calculating RPKM values by VisR^{104 (link)}.