title: GSM4492090: Im-GCT-4072__E3__p5__G34W-repair__H3K36me2; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP-seq was performed as previously described5. Briefly, 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 60 cycles, 10s on 20s off. Samples were checked for sonication efficiency using the criteria of 150–500 bp by gel electrophoresis. After 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.  ChIP reaction for histone modifications was performed on a Diagenode SX-8G IP-Star Compact using Diagenode automated Ideal ChIP-seq Kit. Twenty-five microliter Protein A beads (Invitrogen) were washed and then incubated with antibodies (anti-H3K27me3 (1:40, CST 9733), (anti-H3K27ac (1:80, Diagenode C15410196)), (anti-H3K36me3 (1:100, Active Motif 61021)), (anti-H3.3 (1:66, Millipore 09–838)), (anti-H3.3G34W (1:100, RevMAb 31-1145-00)), (anti-H3K36me2 (1:50, CST 2901)), (anti-H3K9me3 (1:66, Abcam 8898)), and 2 million cells of sonicated cell lysate combined with protease inhibitors for 10 h, followed by 20 min wash cycle with provided wash buffers.   Reverse cross linking took place on a heat block at 65°C for 4 h. ChIP samples were then treated with 2 µl RNase Cocktail at 65°C for 30 min followed by 2 µl 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, following manufacturer's instructions. Briefly, 25 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: 12 cycles of PCR amplification. Size selection was performed after PCR using a 0.6 × /0.8x ratio of Ampure XP beads (double size selection) set to collect 250–450 bp fragments. Single end sequencing was performed on the Illumina HiSeq 2500/4000 platforms.
identifiers.namespace: GEO
identifiers.id: GSM4492090