title: GSM4799147: hct116_t7_chip_rep_1; Homo sapiens; ChIP-Seq
library_construction_protocol: 1x107 cells were harvested, washed and crosslinked with 1% methanol-free formaldehyde in PBS for 8 min at room temperature. Crosslinked cells were lysed for 10 min on ice in 50 l 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. As prolonged sonication caused T7-DNMT3B degradation, chromatin was fragmented using Benzonase as recommended by Pchelintsev et al. 2016 (PMID: 26821228). Briefly, samples were sonicated on ice with Soniprep 150 twice for 30 sec to break up nuclei. Then 200U of Benzonase Nuclease (Sigma) and MgCl2 (final concentration 2.5 mM) were added and samples were incubated on ice for 15 min. The reaction was blocked by adding 10ul of 0.5M EDTA pH 8. 20ug of Spike-in chromatin (ActiveMotif 53083) was added to each sample prior to sonication. 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 T7-Tag antibody (D9E1X, Cell Signalling) in blocking solution (1xPBS, 0.5% BSA) were added and the samples incubated overnight under rotation at 4 oC. 2ul of spike-in antibody per sample (ActiveMotif 61686) was also added in a ratio 1:5 versus the T7-antibody. 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. Libraries were sequenced using the NextSeq 500/550 high-output version 2.5 kit (75bp paired 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: GSM4799147