GSM9474716: HDMB03 H3K27ac HiChIP; Homo sapiens; OTHER
Chromatin crosslinking was conducted using a final concentration of 2% formaldehyde (Sigma Aldrich, F8775) for 10 minutes and was quenched with Arima Stop Solution 1 at a final concentration of 8.7%. The assay was performed on four million HDMB03-Cas12 or D458-Cas9 cells and on three million D425-Cas9 cells. HiChIP data were generated using the Arima-HiC+ kit (P/N A101020), according to the manufacturer's protocol. Chromatin was sheared using the Covaris M220 focused ultrasonicator (PIP: 75 W, DF: 10 %, CPB: 200, Treatment time: 240 to 260 seconds, Setpoint Temperature: 7 °C) in 130 μL cold CS Buffer (10 mM Tris-HCl, pH 8.0, 0.1% SDS). Sheared chromatin was precleared at 4 °C for 1 hour on a rotator by adding 880 μL of cold MR1 Buffer (10 mM Tris-HCL, pH 8.0, 159 mM NaCl, 1.14 mM EDTA, 1.14% Triton X-100, 0.1% SDS, 0.114% Sodium Deoxycholate, 1.14% Protease Inhibitor Cocktail) and 30μL of Protein A Beads (Invitrogen, 10002D). Subsequently, each sample was incubated at 4 °C overnight on a rotator with the H3K27ac antibody (Active Motif, #91193) in an antibody:shearing yield ratio of 0.2. The shearing yield concentration was calculated from 10 µL of the sample taken after chromatin shearing. This aliquot worked as an input control in subsequent analysis. Thereafter, each sample was incubated at 4 °C for 4 hours on a rotator with Protein A Beads (Invitrogen, 10002D) previously overnight blocked with R2 Buffer (10 mM Tris-HCL, pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% Sodium Deoxycholate, 1% Protease Inhibitor Cocktail, 5 mg/mL BSA). Bead-bound enriched chromatin underwent several washes with different buffers to remove nonspecific fragments on a thermomixer at 4 °C for 3 minutes with 1000 rpm shaking. Respectively, three R1 Buffer washes (10 mM Tris-HCL, pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% Sodium Deoxycholate, 1% Protease Inhibitor Cocktail), two R3 Buffer washes (10 mM Tris-HCL, pH 8.0, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% Sodium Deoxycholate), one LC Buffer wash (10 mM Tris-HCL, pH 8.0, 150 mM Lithium Chloride, 1 mM EDTA, 0.5% IGEPAL CO-630, 0.1% Sodium Deoxycholate) and two LTE Buffer washes (10 mM Tris-HCL, pH 8.0, 0.1 mM EDTA). Reverse crosslinking followed the manufacturer's protocol. Biotin enrichment was carried out using 100μL of Arima Enrichment Beads prior to library construction. Libraries were prepared as described in the Arima-HiC+ HiChIP Library Preparation protocol, using Swift Accel-NGS® 2S Plus DNA Library Kit (Integrated DNA Technologies, 10009878), KAPA Library Amplification Kit (Roche Sequencing Solutions, 26148) and Swift Single Indexes, with 10 to 13 amplification cycles. Libraries were sequenced as 151 bp paired end reads on the Illumina NovaSeq X Plus system.