GSM6209069: BT245, KO, inputForK4me3; Homo sapiens; ChIP-Seq
Cells (cell lines or dissociated tumor cells) were fixed with 1% formaldehyde (Sigma). Fixed cell preparations were washed, pelleted and stored at -80°C. Sonication of lysed nuclei (lysed in a buffer containing 1% SDS) was performed on a BioRuptor UCD-300 for 18-30 cycles, 30s on 30s off, centrifuged every 15 cycles, chilled by 4°C water cooler. Samples were checked for sonication efficiency using the criteria of 150-500bp by gel electrophoresis. After the sonication, the chromatin was diluted to reduce SDS level to 0.1% and before ChIP reaction 2% of sonicated drosophila S2 cell chromatin was spiked-in the samples for quantification of total levels of histone mark after the sequencing (see below).  ChIP reaction for histone modifications was performed on a Diagenode SX-8G IP-Star Compact using Diagenode automated Ideal ChIP-seq Kit. 25ul Dynabeads Protein A beads (Invitrogen) Dynabaeds M-280 Sheep anti-Mouse IgG beads (Invitrogen) were washed and then incubated with antibodies (anti-H3K27me3 (1:40, CST 9733), anti-H3K4me3 (1:40, CST 9751), anti-H2AK119ub (1:40, CST 8240), anti-H3K27ac (1:100, Diagenode C15410196), anti-H3K36me2 (1:50, CST 2901)). One-two million cells of sonicated cell lysate combined with protease inhibitors for 10 hr, followed by 20 min wash cycle with provided wash buffers.   ChIP reactions for SUZ12, RING1B, CBX2 and CTCF were performed as follows: antibodies (anti-SUZ12 (1:150, CST 3737), anti-RING1B (1:200, Active Motif 39663), anti-CBX2 (Bethyl A302-524 1:200), anti-CTCF (1:400, Diagenode C15410210)) were conjugated by incubating with 40ul protein A or G beads at 4°C for 6 hours, then chromatin from 5-10 million cells was added in RIPA buffer, incubated at 4°C o/n, washed using buffers from Ideal ChIP-seq Kit (1 wash with each buffer, corresponding to RIPA, RIPA+500mM NaCl, LiCl, TE), eluted from beads by incubating with Elution buffer for 30 minutes at room temperature.   Reverse cross linking took place on a heat block at 65°C for 4 hr. ChIP samples were then treated with 2ul RNase Cocktail at 65°C for 30 min followed by 2ul Proteinase K at 65°C for 30 min. Samples were then purified with QIAGEN MiniElute PCR purification kit as per manufacturers' protocol. In parallel, input samples (chromatin from about 50,000 cells) were reverse crosslinked, and DNA was isolated following the same protocol. Library preparation was carried out using Kapa HTP Illumina library preparation reagents. Briefly, 25ul of ChIP sample was incubated with 45ul end repair mix at 20°C for 30 min followed by Ampure XP bead purification. A tailing: bead bound sample was incubated with 50ul buffer enzyme mix for 30°C 30 min, followed by PEG/NaCl purification. Adaptor ligation: bead bound sample was incubated with 45ul buffer enzyme mix and 5ul of different TruSeq DNA adapters (Illumina) for each sample, for 20°C 15 min, followed by PEG/NaCl purification (twice). Library enrichment: 12 cycles of PCR amplification. Size selection was performed after PCR using a 0.6x/0.8x ratio of Ampure XP beads (double size selection) set to collect 250-450bp fragments.