GSM8078725: HiChIP_ES_WT_SRF; Mus musculus; OTHER
HiChIP was performed as described previously [Mumbach, 2016] with the following modifications. 5e107 ESCs were trypsinized, pelleted by centrifugation, resuspended in 20 ml 1% formaldehyde and incubated at room temperature for 10 minutes. 2 ml 2.5 M glycine was added followed by 5 minute incubation and centrifugation. The pelleted cells were lysed in 2.1 ml HiC lysis buffer (LB) (10 mM Tris pH8, 10 mM NaCl, 0.2% NP-40, protease inhibitors), split equally in 3x 1.5 ml eppendorf tubes, rotated at 4°C for 30 minutes, and centrifuged at 2,500xg for 5 minutes. Each pellet was once again resuspended in 700 μl HiC LB, centrifuged, resuspended in 100 μl 0.5% SDS and incubated at 62°C for 10 minutes. 285 μl H2O and 50 μl 10% TX-100 were added followed by incubation at 37°C for 15 minutes. For restriction digestion, 15 μl (375 units) MboI (NEB, R0147M) and 50 μl 10x NEBuffer 2.1 were added, and incubated at 35°C for 9 hours. MboI was then heat inactivated at 62°C for 20 minutes. For biotin incorporation and proximity ligation, 52 μl of the mix containing 37.5 μl 0.4 mM biotin-14-dATP (ThermoFisher, 19524016), 1.5 μl 10 mM dTTP, 1.5 μl 10 mM dCTP, 1.5 μl 10 mM dGTP, 10 μl 5 units/μl DNA Pol. I, Large (Klenow) Fragment (NEB, M0210L) were added and rotated at 37°C for 90 minutes. 948 μl ligation master mix (150 μl 10x T4 DNA Ligase Reaction buffer (NEB, B0202S), 125 μl 10% TX-100, 3 μl 50 mg/ml BSA, 10 μl 400 units/μl T4 DNA ligase (NEB, M0202S), 660 μl H2O) were added and incubated at room temperature for 5 hours. The tubes were the then centrifuged and the pellets resuspended in 1 ml sonication buffer (10 mM Tris pH 8, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine), transferred to 15 ml Falcon tubes and sonicated using a W-450D Digital Sonifier sonicator for 18 cycles of 10 seconds on/50 seconds off with Amplitude set at 18 in a 4°C room with tubes chilled in ice water. After sonication, 110 μl of 10% Triton X-100 were added per tube, transferred to two 1.5 ml Eppendorf tubes and debris was pelleted by centrifugation at 20,000g and 4°C for 10 minutes. For ChIP, 3.3 ml of the combined cleared samples were mixed with 1.5 ml of dilution buffer (16.7 mM Tris pH 7.5, 167 mM NaCl, 1.1% Triton X-100, 1.2 mM EDTA) containing protease inhibitors. In parallel with biotin incorporation, 150 μl protein G-coated Dynabeads were washed 3x with 1 ml of blocking buffer (PBS, 0.5% BSA), resuspended in 500 μl of blocking buffer containing 12 μg SRF ChIP antibody and rotated at 4°C for 10 hours, followed by 3x washing with 1 ml blocking buffer. 4.8 ml cleared sonicated samples were added to 150 μl beads pre-incubated with the SRF antibody and rotated overnight at 4°C. The following day, 5 washing steps with 1 ml RIPA buffer (50 mM Hepes pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate) and one washing step with 1 ml of TEN (10 mM Tris pH 8, 1 mM EDTA, 50 mM NaCl) were performed. The elution was performed in two subsequent steps using 150 μl of 1x elution buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS) and incubation at 65°C while shaking for 10 minutes each. 300 μl of TE buffer were added to the combined eluates, and RNA was digested using 0.2 μg/ml RNase A at 37°C for 30 minutes. Proteins were digested with the addition of 0.2 μg/ml proteinase K and incubation at 55°C for 45 minutes. Crosslinking was reversed by incubation at 65°C for 4 hours. The post-ChIP DNA was purified using MinElute (Qiagen) and quantified using the Qubit DNA HS assay. For biotin pull-down, 5 μl of MyOne Streptavidin C1 Dynabeads (ThermoFisher, 65001) were washed with 1 ml Tween wash buffer (5 mM Tris pH 7.5, 0.5 mM EDTA, 1M NaCl, 0.05% Tween-20), resuspended in 10 μl 2x Biotin binding buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2M NaCl), combined with 50 ng post-ChIP DNA in 10 μl of H2O, and incubated at room temperature for 15 minutes. The beads were then washed twice with 500 μl Tween wash buffer at 55°C for 2 minutes each and resuspended in 50 μl of the transposition reaction mix (25 μl 2x TD buffer, 2.5 μl Tn5 transposase, and 22.5 μl H2O) followed by a 9 minute incubation at 55°C. The beads were then incubated in 50 mM EDTA at 50°C for 30 minutes, washed twice with 500 μl of 50 mM EDTA at 50°C for 3 minutes, twice with 500 μl of Tween wash buffer at 55°C for 2 minutes, and once with 500 μl of 10 mM Tris pH 7.5. The beads were then resuspended in 50 μl of master mix (25 μl 2x Kapa HiFi Hotstart Ready Mix, 1.25 μl of 10 μM Nextera Ad1.1, 1.25 μl of 10 μM Nextera Ad2.2, 22.5 μl H2O) and amplification was performed for 45 seconds at 98°C, 8 cycles of [15 seconds at 98°C, 30 seconds at 63°C and 45 seconds at 72°C] and a final 1 minute incubation at 72°C. After PCR, 46 μl of the library were transferred to a new tube. 38 μl of AMPure XP beads (Beckman Coulter) were used for a single purification and the DNA was eluted using 10 μl of elution buffer. The library was quantified using the Qubit DNA HS assay and the library size was validated using DNA HS Bioanalyzer chips.