GSM8999227: HCT116_h3k36me2_chip_rep_2_chip_seq; Homo sapiens; ChIP-Seq
For H3K36me2, native ChIP-seq was performed adapting ultra-low-input (ULI) ChIP (Brind'Amour & Lorincz, 2022) as follows. Nuclei were isolated from 1x106 unfixed cells using PBS 0.1% NP-40 and resuspended in 75 μl of Nuclear Isolation buffer (10mM Tris HCl, 0.1% NaDeOx, 0.1% Triton-100) and 75 μl of 2X MNase digestion Buffer (NEB). Chromatin was digested using MNase (NEB) for 6 min at 37 °C. Enzyme concentration was determined experimentally to obtain a majority of mononucleosomes and reaction stopped adding 5 μl of 0.5 M EDTA pH 8. Nuclei were lysed as follows: 30 μl of 5X Nuclear Lysis Buffer (5% NaDeOx, 5% Triton X-100) were added and samples vortexed, then 160 μl of ChIP Dilution buffer (0.1% Triton X-100, 20 mM Tris-HCl (pH 8.1), 150mM NaCl) were added and samples sonicated using Bioruptor Plus sonicator (Diagenode) for two cycles (30 sec on/30 sec off on high setting at 4 °C). Following centrifugation for 30 min at maximum speed at 4 °C, supernatants were collected and 350 μl of ChIP Dilution buffer added. Half the sample was retained to check chromatin fragmentation and half processed for immunoprecipitation as previously described. 10% of input retained and antibody-bound-beads added for overnight incubation at 4 °C. 3 μl of anti-H3K36me2 (Invitrogen, T.571.7, MA5-14867) were used per sample. 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). ChIP-seq libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and NEBNext Multiplex Oligos for Illumina (NEB) barcode adapters according to the manufacturer instructions. were used. Specifically, Illumina Index Primers Set 1 (E7335) for 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. H3K36me3 ChIP-Rx-seq libraries were sequenced using the NextSeq 500/550 high-output version 2.5 kit (75 bp single end reads). 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 at the Edinburgh Clinical Research Facility.