title: GSM6578716: ChIP_EZH2_WT_rep1; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were washed once with ice-cold PBS and pelleted at 1000 g for 5 min at 4°C. Crosslinked cells were resuspended in ice-cold hypotonic buffer, and centrifuged at 1000 g for 5 min at 4°C to obtain a nuclear fraction. Nuclear pellets were resuspended in  either LB3 buffer for H3K27me3 (Active motif, 39155) or PIPA-NR buffer for SUZ12 (Abcam, ab12073) and EZH2 (Invitrogen, 49-1043). Chromatin DNA was sonicated in a 96 Place microTUBE Rack (Covaris) using a Covaris E220 for 12-18 cycles. Samples were centrifuged at 15000 rpm for 10 min at 4°C, and the supernatant was used for immunoprecipitation. LB3 lysate was diluted 1.1-fold with 10% Triton X-100, and 1% of the lysate was kept as ChIP input. The immunoprecipitated chromatin samples were used for library preparation with NEBNext Ultra II Library kit (NEB) according to the manufacturer's instructions. DNA was purified and eluted following by amplification for 16 cycles PCR reactions using NEBNext High-Fidelity 2X PCR Master Mix and 0.5 mM each of primers Solexa 1GA and Solexa 1GB. The amplified libraries were purified, eluted and size selected using PAGE/TBE gel for 225-325 bp fragments by gel extraction, and single-end sequenced on NovaSeq 6000 (Illumina). The eluted input DNA was prepared for libraries as described for ChIP samples.