Chromatin immunoprecipitation sequencing (ChIP-seq)

Native ChIP-seq for H3K27me3, H3K36me2 and H3K36me3 was performed as described previous(Karimi et al., 2011) with minor adjustments. Briefly ~80–200 pairs of eye disc tissue were dissected from H3.3 WT, K27M, K36M, and negative control yw flies and flash frozen. Tissue was disassociated passing through a syringe (25 5/8 gauge) 20x in douncing buffer (10mM Tris Cl pH7.5, 4mM MgCl₂, 1mM CaCl₂, and protease inhibitor cocktail (Roche)). Disassociated cells were then digested with 30U of micrococcal nuclease (Worthington) at 37°C for 7 mins, yielding predominantly mono- and di-nucleosomes. Cells were then lysed in ice-cold hypotonic lysis buffer (0.2 mM EDTA, 0.1 mM benzamidine, 0.1 mM PMSF, 1.5 mM DTT, and protease inhibitor cocktail (Roche)) to yield chromatin. Chromatin was then incubated with pre-conjugated antibody:bead complex containing H3K27me3 (CST 9733, 3 μL / IP), H3K36me2 (Active Motif 39255, 3 μL / IP), H3K36me3 (Active Motif 61022, 3 μL / IP) complexed to Protein A Dynabeads (Invitrogen, 20 μL / IP) or anti-mouse IgG Dynabeads (Invitrogen, 20 μL / IP) rotating at 4°C overnight. Beads were then washed 2x in ChIP Wash Buffer (20 mM Tris-HCl pH 8, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, and protease inhibitor cocktail) and 1x in Final Wash Buffer (20 mM Tris-HCl pH 8, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, and protease inhibitor cocktail). DNA was then eluted in 100mM NaHCO₃ and 1% SDS at 65°C for 2h, and purified using phenol:chloroform extraction followed by ethanol precipitation.

Library preparation was carried out using KAPA HTP Illumina library preparation reagents, following manufacturer’s instructions. Briefly, 50 µl of ChIP sample was incubated with 45 µl end repair mix at 20 °C for 30 min followed by Ampure XP bead purification. A tailing: bead bound sample was incubated with 50 µl buffer enzyme mix at 30 °C for 30 min, followed by PEG/NaCl purification. Adaptor ligation: bead-bound sample was incubated with 45 µl buffer enzyme mix and 5 µl of TruSeq DNA adapters (Illumina), for 20 °C 15 min, followed by PEG/NaCl purification (twice). Library enrichment: 14 cycles of PCR amplification. Size selection was performed after PCR using gel excision to collect 150–450 bp fragments. ChIP libraries were sequenced on Illumina NovaSeq 6000 platforms at 50 bp paired-reads.