ChIP was performed as described previously (40). Briefly, H3.3K27M was induced by doxycycline (1 μg/ml) for 72 hours, cross-linked with 1% formaldehyde, and harvested following glycine treatment to quench the formaldehyde. Nuclei were extracted, sonicated using the Diagenode Bioruptor to produce chromatin fragments at an average size of 300 to 500 bp. Chromatin was then precleared with bovine serum albumin (BSA)–blocked magnetic Dynabeads. Each ChIP was performed with an internal spike-in standard corresponding to 1:200 of cross-linked, sonicated, and precleared Drosophila chromatin. The Drosophila-specific H2A.V antibody was also added at ~1:20 of the primary ChIP antibody. The immunoprecipitation was performed using antibodies to the appropriate protein (table S2) and 30 μl of BSA-blocked Dynabeads. Immunoprecipitated chromatin bound to the beads was then washed in radioimmunoprecipitation assay buffer. Chromatin was de–cross-linked, and proteins were removed by proteinase K. DNA libraries were constructed, barcoded as in (17), and subjected to SR50 sequencing on an Illumina HiSeq 2500 or Illumina NextSeq 500.

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