title: GSM4799144: hct116_dko_k36me3_chip_rep_2; Homo sapiens; ChIP-Seq
library_construction_protocol: 0.5x107 cells were harvested and crosslinked with 1% methanol-free formaldehyde in PBS for 5 min at room temperature. Crosslinked Drosophila S2 cells were spiked into samples before sonication at a ratio of 20:1 human to Drosophila cells. Following nuclei rupture by sonication on ice with Soniprep 150, chromatin was fragmented using Bioruptor Plus sonicator (Diagenode) for 40 cycle (30 s on/30 s off on high setting at 4oC). Crosslinked cells were lysed for 10 min on ice in 50ul of lysis buffer (50mM Tris-HCl pH8, 150mM NaCl, 1mM EDTA, 1% SDS) freshly supplemented with proteinase inhibitor (Sigma-Aldrich). IP dilution buffer (20 mM Tris-HCl pH8, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100) freshly supplemented with proteinase inhibitor, DTT and PMSF was added to the samples to reach a final volume of 500ul. Following centrifugation for 30 min at 14,000 rpm at 4 oC, supernatants were collected and supplemented with Triton X-100 (final concentration 1%) and 5% input aliquots were retained for later use. Protein A dynabeads (Invitrogen) previously coupled with H3K36me3 antibody (ab9050, Abcam) in blocking solution (1xPBS, 0.5% BSA) were added and the samples incubated overnight under rotation at 4 oC. Beads were then washed for 10 min at 4 oC with the following buffers: IP dilution buffer 1% Triton X-100 (20 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100), buffer A (50 mM Hepes pH 7.9, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS), buffer B (20 mM Tris pH 8, 1mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate), TE buffer (1 mM EDTA pH 8, 10 mM Tris pH 8). Chromatin was eluted by incubating the beads in extraction buffer (0.1M NaHCO3, 1% SDS) for 15 min at 37oC. To reverse the cross-linking Tris-HCl pH 6.8 and NaCl were added to final concentrations of 130 mM and 300mM respectively. IP samples were then incubated at 65 oC overnight. Samples were then incubated at 37 oC for 1h after addition of 2ul of RNase Cocktail Enzyme Mix (Ambion). Then 40 ug of Proteinase K (Roche) were added, followed by 2h incubation at 55oC. Input material was similarly de-crosslinked. Samples were purified with the MinElute PCR purification kit (QIAGEN). Libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (E7645) according to the manufacturer instructions. Barcoded adapters (NEBNext® Multiplex Oligos for Illumina® Index Primers Set 1, E7335) were used. Adapter-ligated DNA was size selected for an insert size of 150 bp using AMPure XP beads.  Libraries were sequenced using the NextSeq 500/550 high-output version 2.5 kit (75bp single end reads). Libraries were combined into equimolar pools to run within individual flow cells. Sequencing was performed by the Edinburgh Clinical Research Facility.
attributes.tag: GEO Accession
attributes.value: GSM4799144