GSM7818426: K27ac-MEF-KDM6A-WT; Mus musculus; ChIP-Seq
For crosslinking, cells were pelleted and resuspended in PBS containing 1% fresh formaldehyde for 8 min at room temperature with rotation. Crosslinking was quenched by addition of 1/10 volume of 1.25M glycine and incubated for 5 minutes at room temperature with rotation. For isolation of nuclei, cell pellets were washed once in ice-cold PBS and then resuspended in lysis buffer (10% glycerol, 1% Triton TX-100, 0.1 mM EDTA, 14mM NaCl 25mM Hepes pH 7.5) and incubated for 5 minutes on ice. Cell nuclei were pelleted and incubated 5 min in wash buffer (0.1 mM EDTA, 20mM NaCl, 2mM Tris-HCl pH 7.5). Cell nuclei were pelleted and resuspended in 1 ml sonication buffer (1 mM EDTA, 10mM Tris-HCl pH 7.5) at a concentration equivalent to 10x106 cells per ml. Samples were sonicated in 1ml in AFA fiber Covaris tubes using the Covaris E220 (Woburn, MA) for 30 minutes using the following settings: 15% Duty Cycle, 200 cycles/burst, and Peak incidence 140. Prior to immunoprecipitation, sheared chromatin was diluted 1:1 in 2X ChIP dilution buffer (2% Triton X-100, 2mM EDTA, 300mM NaCl, 30mM Tris-HCl pH 8). Sheared chromatin samples were block with 20 µl Protein A/G Dyna beads (Invitrogen) for 2h at 4°C with rotation. Immunoprecipitations were performed overnight (16 hours, 4°C, with rotation) with the following antibodies: H3K27Ac (Acitve Motif #39133), H3K27me3 (Cell Signaling Technologies #9733), KDM6A (Cell Signaling Technologies #33510). Samples were washed once with ChIP dilution buffer(1% Triton X-100, 1mM EDTA, 150mM NaCl, 15mM Tris-HCl pH 8), low salt wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 100mM NaCl, 20mM Tris-HCl pH 8), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 500mM NaCl, 20mM Tris-HCl pH 8), LiCl wash buffer (1% deoxycholate, 1% NP-40, 2mM EDTA, 250mM LiCl, 20mM Tris-HCl pH 8), and Tris buffer (10mM Tris-HCl pH 7.5). For library construction, beads were incubated in 15 µl Tagmentase buffer with 1µl Tagment enzyme (Illumina Nextera DNA library Prep kit #15028212) and incubated at 37°C for 10 min. Immediately after, beads were washed once in 1ml ChIP dilution buffer and in 1 ml TE (10mM Tris-HCl pH 7.5, EDTA 1mM) prior to incubation in reverse crosslinking buffer (300mM NaCl, 10 mM EDTA, 0.5% SDS, 20mM Tris-HCl pH 8) for 4 hours at 65°C with shaking. Finally, proteinase K digestion was performed for 1h at 55°C with shaking prior to DNA purification using Zymogen ChIP DNA Clean and Concentrator Kit (Zymo Research, D5205, Irvine, CA). Libraries were generated by amplification of ChIP DNA using KAPA HiFi Hotstart Library Amp kit (Roche #07959028001, Indianapolis, IN) and size selected for between 200 bp and 700 bp using Ampure beads XP (Beckman Coulter, Brea, CA). Library were quantified and QC using D1000 high-sensitivity screen tape with 4200 TapeStation Instrument (Agilent Technologies, Santa Clara, CA) and sequenced on an Ilumina Hi-Seq 3000 with 150 bp pair-end reads.