Instrument: Illumina NovaSeq 6000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Lysates were cleared by centrifugation and 1/20th input was saved. H3K27me2/3 antibody was added (Active Motif, 2uL) and samples were rotated overnight at 4oC. The next day, equilibrated Protein A/G agarose (Santa Cruz Biotechnology) was added to bind the antibody, incubated for 3 hours at 4oC, and washed twice in cold ChIP Lysis buffer, once with ChIP Lysis buffer + 0.5M NaCl, once with LiCl wash buffer (10mM Tris pH 8.0, 250mM LiCl, 1mM EDTA, 0.5% NP40), and DNA/protein was eluted with TES buffer (50mM Tris pH 8.0, 10mM EDTA, 1% SDS) and incubated at 65oC. Samples (IP and input) were decrosslinked at 65oC overnight. The next day, samples were proteinase K treated for 2 hours at 50oC, and DNA was purified using the Qiagen MinElute PCR purification kit and eluted in 30 μl of water. Approximately 10 ng of DNA was used to generate ChIP-Seq libraries. Each library was prepared using the NEB Next DNALibrary Prep Kit for Illumina according to the manufacturer's instructions. AMPure XP beads (Beckman Coulter A63881) were used for size selection of ~200-800 bp fragments. Final libraries were PCR-amplified using one cycle at 98 °C for 30 sec, 10 cycles at 98 °C for 10 sec, 60 °C for 30 sec and 72 °C for 30 sec and a final extension at 72 °C for 5 min.