GSM7884911: 3BKO_T73BW263A_chip_rep_2_ChIPseq; Homo sapiens; ChIP-Seq
ChIP-seq was performed as previously described (Masalmeh et al., 2021, PMID: 33514701). For T7-DNMT3B ChIP-Rx-seq experiments, 1×107 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 μl of lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1% SDS) freshly supplemented with proteinase inhibitor (Sigma-Aldrich). IP dilution buffer (20 mM Tris-HCl pH 8, 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 500 μl. As prolonged sonication caused T7-DNMT3B degradation, chromatin was fragmented using Benzonase (Pchelintsev et al., 2016, PMID: 26821228): samples were sonicated on ice with Soniprep 150 twice for 30 s to break up nuclei; then 200 U 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 10 μl of 0.5 M EDTA pH 8. Following centrifugation for 30 min at 18,407 g at 4 °C, 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 10 μl of T7-Tag antibody per 1×107 cells in blocking solution (1x PBS, 0.5% BSA) were added and the samples incubated overnight under rotation at 4 °C. Beads were then washed for 10 min at 4 °C 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 (50mM 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, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate), TE buffer (1mM EDTA pH 8, 10 mM Tris pH 8). Chromatin was eluted by incubating the beads in extraction buffer (0.1 M NaHCO3, 1% SDS) for 15 min at 37 °C. To reverse the cross-linking Tris-HCl pH 6.8 and NaCl were added to final concentrations of 130 mM and 300 mM respectively, and immunoprecipitations were incubated at 65 °C overnight. Samples were then incubated at 37 °C for 1 h after addition of 2 μl of RNase Cocktail Enzyme Mix (Ambion). Then 40 μg of Proteinase K (Roche) were added, followed by 2 h incubation at 55 °C. Input material was similarly de-crosslinked. Samples were purified with the MinElute PCR purification kit (QIAGEN). For ChIP-Rx-seq of endogenous T7-DNMT3B and ectopic T7-DNMT3B expressed in DNMT3B KO cells, 20 μg of Spike-in chromatin (ActiveMotif 53083) was added to each sample after sonication. 2 μl of spike-in antibody per sample (ActiveMotif 61686) was also added in a ratio 1:5 versus the T7 antibody. A similar protocol was used for H3K4me3, H3K9me3, H3K27me3 and H3K36me3 ChIP-seq experiments, except: 0.5×107 cells were harvested and crosslinked with 1% methanol-free formaldehyde in PBS for 5 min at room temperature. For H3K36me3 ChIP-Rx-seq 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 cycles (30 s on/30 s off on high setting at 4 °C). 2 μl /1×106 cells of the following antibodies were used for immunoprecipitations: H3K4me3 (EpiCypher 13-00041), H3K9me3 (Active Motif 39161), H3K27me3 (Cell Signaling Technology C36B11) and H3K36me3 (Abcam ab9050). Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645) according to the manufacturer instructions. NEBNext Multiplex Oligos for Illumina (NEB) barcode adapters were used. Specifically, Illumina Index Primers Set 1 (E7335) for endogenous T7-DNMT3B and H3K36me3, and Unique Dual Index UMI Adaptors DNA Set 1 (E7395) for the other libraries.  For histone modifications ChIP-seq, adapter-ligated DNA was size selected for an insert size of 150 bp using Agencourt AMPure XP beads. Libraries were quantified using the Qubit dsDNA HS or BR assay kit and assessed for size and quality using the Agilent Bioanalyser. Endogenous T7-DNMT3B and H3K36me3 ChIP-Rx-seq libraries were sequenced using the NextSeq 500/550 high-output version 2.5 kit (75 bp paired end reads, DNMT3B or 75 bp single end reads, H3K36me3). The other ChIP-seq libraries were sequenced using the NextSeq 2000 P3 (50 bp paired end reads). Libraries were combined into equimolar pools to run within individual flow cells. Sequencing was performed by the Edinburgh Clinical Research Facility.