GSM6235195: S9CC13_R4_dTAG0h_K27ac; Homo sapiens; ChIP-Seq
One fully confluent 10 cm plate of cells was cross-linked per ChIP experiment in 10 mL PBS with 1% methanol-free formaldehyde for 10 min and quenched with a final concentration of 0.125M glycine for 5 min with nutation. Cross-linked cells were scraped into tubes with 0.001% Triton X in PBS, washed with PBS without Triton, pelleted by centrifugation, flash-frozen in liquid nitrogen and stored at −80°C. Samples were defrosted on ice and resuspended in 5mL LB1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, with 1X cOmplete Protease Inhibitor Cocktail and PMSF) and rotated vertically for 10 min at 4°C. Samples were centrifuged for 5 min at 1350 x g at 4°C, and resuspended in 5mL LB2 (10 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, with 1X cOmplete Protease Inhibitor Cocktail and optionally 1mM PMSF) and rotated vertically for 10 min at 4°C. Samples were centrifuged for 5 min at 1350 x g at 4°C, resuspended in 300 uL LB3 per sonicated sample, and incubated for 10 min on ice. Samples were sonicated in 1.5 mL Bioruptor Plus TPX microtubes (Diagenode, c30010010-50) on Bioruptor Plus for 10 cycles of 30sec ON/30sec OFF. Every 5 cycles, samples were lightly vortex and briefly centrifuged. Samples were diluted in additional LB3 to 1 mL, pelleted at 16,000 RCF for 10 min, and the supernatant was removed. Triton X-100 was added to 1%. To check DNA size distribution and quantity, a 10 uL aliquot of sonicated chromatin from each sample was diluted to 100 uL in Elution Buffer (50 mM Tris, 10 mM EDTA, 1% SDS) with 0.0125 M NaCl and 0.2 mg/mL RNase A and incubated at 65C for 1 hour, followed by addition of Proteinase K to 0.2 mg/mL and an additional 1 hour of 65C incubation. DNA was purified using Zymo DNA Clean & Concentrator Kit with ChIP DNA Binding Buffer (Zymo, D5201-1-50) and size distribution and quantity was assessed by separation on a 1% agarose gel and Qubit HS DNA kit, respectively. Qubit measurements were used to normalize samples to the same DNA concentration. Following normalization, the chromatin was divided for input (2%) and ChIP samples. A minimum of 25 ug DNA was used for histone ChIPs, and 50 ug for V5 ChIPs. 5 μg anti-H3K27ac (Active Motif, 39133) antibody or 10 μg anti-V5 (Abcam, ab9116) antibody was added per ChIP sample, and incubated overnight at 4°C. Protein G Dynabeads (ThermoFisher) were first blocked with Block solution (0.5% BSA (w/v) in 1X PBS) and then added to cleared chromatin to bind antibody-bound chromatin for a 4-6 hour incubation. Chromatin-bound Dynabeads were washed at least 6 times with chilled RIPA wash buffer (50 mM HEPES-KOH pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate), followed by a wash with chilled TE + 50 mM NaCl. Chromatin was eluted for 30 min in Elution Buffer (50 mM Tris, 10 mM EDTA, 1% SDS) at 65°C with frequent vortexing. The ChIP and input samples were then incubated at 65°C overnight to reverse cross-links (12-16 hours). Samples were diluted and sequentially digested with RNase A (0.2 mg/mL) for 2 hours at 37°C followed by Proteinase K (0.2 mg/mL) for 2 hours at 55°C for 2-4 hours to digest protein. ChIP and input samples were purified by Zymo DNA Clean & Concentrator Kit with ChIP DNA Binding Buffer. Samples were quantified by Qubit dsDNA HS assay kit, and 10-50ng of ChIP DNA was used for library preparation with end repair, A-tailing, and adaptor ligation (NEB). Following USER enzyme treatment, libraries were cleaned up with one round of single-side AMPure XP bead clean-up, then amplified to add indices using NEBNext Ultra II Q5 Master Mix and NEBNext Multiplex Oligos for Illumina kit (NEB, E7335S) with 4-6 cycles (as determined by input amounts from NEB protocol). ChIP libraries were purified by two rounds of double-sided AMPure XP bead clean-up (0.5x then 0.4x initial sample volume of beads added) to remove large fragments and deplete adaptors. Library concentration and quality within ChIP or input groups was assessed by Qubit dsDNA HS assay kit and separation on a PAGE gel, and used to pool within ChIP or input groups. KAPA qPCR was used to pool across ChIP or input groups. Pooled libraries were sequenced using Novaseq 6000 platform (2x 150bp).