GSM5059681: HiChIP SMC1A A673 STAG2 WT Replicate 1; Homo sapiens; OTHER
HiChIP was performed on the A673 clonal lines sgNT-1c4 and sgSTAG2-1c6 in duplicate based on a previously published protocol (Mumbach et al., 2016) with a few adaptations which were performed as previously described (Weintraub et al., 2017). The A673 clonal lines sgNT-1c4 and sgSTAG2-1c6 were tested with duplicates for each line crosslinked on separate occasions. The SMC1A antibody used in the ChIP experiments was by Bethyl A300-055A. Libraries were sequenced 100x100 on an Illumina Hi-Seq 2500 platform. HiChIP was performed as described in (Mumbach et al., 2016) with a few modifications. Ten million cells were cross-linked for 10 min at room temperature with 1% formaldehyde in growth media and quenched in 0.125 M glycine. After washing twice with ice-cold PBS, the supernatant was aspirated and the cell pellet was flash frozen in liquid nitrogen and stored at -80°C. Cross-linked cell pellets were thawed on ice, resuspended in 800 µL of ice-cold Hi-C lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, and 0.2% IGEPAL CA-630 with 1× cOmplete protease inhibitor (Roche, 11697498001)), and incubated at 4°C for 30 minutes with rotation. Nuclei were pelleted by centrifugation at 2500 rcf. for 5 min at 4°C and washed once with 500 µL of ice-cold Hi-C lysis buffer. After removing supernatant, nuclei were resuspended in 100 µL of 0.5% SDS and incubated at 62°C for 10 minutes. SDS was quenched by adding 335 µL of 1.5% Triton X-100 and incubating for 15 minutes at 37°C. After the addition of 50 µL of 10X NEB Buffer 2 (NEB, B7002) and 375 U of MboI restriction enzyme (NEB, R0147), chromatin was digested at 37°C for 2 hours with rotation. Following digestion, MboI enzyme was heat inactivated by incubating the nuclei at 62°C for 20 min. To fill in the restriction fragment overhangs and mark the DNA ends with biotin, 52 µL of fill-in master mix, containing 37.5 µL of 0.4 mM biotin-dATP (Invitrogen, 19524016), 1.5 µL of 10 mM dCTP (Invitrogen, 18253013), 1.5 µL of 10 mM dGTP (Invitrogen, 18254011), 1.5 µL of 10 mM dTTP (Invitrogen, 18255018), and 10 µL of 5 U/µL DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210), was added and the tubes were incubated at 37°C for 1 hour with rotation. Proximity ligation was performed by addition of 947 µL of ligation master mix, containing 150 µL of 10X NEB T4 DNA ligase buffer (NEB, B0202), 125 µL of 10% Triton X-100, 7.5 µL of 20 mg/mL BSA (NEB, B9000), 10 µL of 400 U/µL T4 DNA ligase (NEB, M0202), and 655.5 µL of water, and incubation at room temperature for 4 hours with rotation. After proximity ligation, nuclei were pelleted by centrifugation at 2500 rcf. for 5 minutes and resuspended in 1 mL of ChIP sonication buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, and 0.1% SDS with protease inhibitor). Nuclei were sonicated using a Covaris S220 for 6 minutes with the following settings: fill level 8, duty cycle 5, peak incidence power 140, cycles per burst 200. Sonicated chromatin was clarified by centrifugation at 16,100 rcf. for 15 min at 4°C and supernatant was transferred to a tube. 60 µL of protein G magnetic beads were washed three times with sonication buffer, resuspended in 50 µL of sonication buffer. Washed beads were then added to the sonicated chromatin and incubated for 1 hour at 4°C with rotation. Beads were then separated on a magnetic stand and the supernatant was transferred to a new tube. 7.5 µg of SMC1A antibody (Abcam, ab4729) or 7.5 µg of YY1 antibody (Abcam, ab109237) was added to the tube and the tube was incubated overnight at 4°C with rotation. For YY1 six reactions were carried out and pooled prior to tagmentation. The next day, 60 µL of protein G magnetic beads were washed three time in 0.5% BSA in PBS and washed once with sonication buffer before being resuspended in 100 µL of sonication buffer and added to each sample tube. Samples were incubated for 2 hours at 4°C with rotation. Beads were then separated on a magnetic stand and washed three times with 1 mL of high salt sonication buffer (50 mM HEPES-KOH pH 7.5, 500 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) followed by three times with 1 mL of LiCl wash buffer (20 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS) and once with 1 mL of TE with salt (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 50 mM NaCl). Beads were then resuspended in 200 µL of elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1% SDS) and incubated at 65°C for 15 minutes to elute. To purify eluted DNA, RNA was degraded by the addition of 2.5 µL of 33 mg/mL RNase A (Sigma, R4642) and incubation at 37°C for 2 hours. Protein was degraded by the addition of 10 µL of 20 mg/mL proteinase K (Invitrogen, 25530049) and incubation at 55°C for 45 minutes. Samples were then incubated at 65°C for 5 hours to reverse crosslinks. DNA was then purified using Zymo DNA Clean and Concentrate 5 columns (Zymo, D4013) according to manufacturer's protocol and eluted in 14 µL water. The amount of eluted DNA was quantified by Qubit dsDNA HS kit (Invitrogen, Q32854). Tagmentation of ChIP DNA was performed using the Illumina Nextera DNA Library Prep Kit (Illumina, FC-121-1030). First, 5 µL of streptavidin C1 magnetic beads (Invitrogen, 65001) was washed with 1 mL of tween wash buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA pH 8.0, 1 M NaCl, 0.05% Tween-20) and resuspended in 10 µL of 2X biotin binding buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 2 M NaCl). 54.19 ng purified DNA was added in a total volume of 10 µL of water to the beads and incubated at room temperature for 15 minutes with agitation every 5 minutes. After capture, beads were separated with a magnet and the supernatant was discarded. Beads were then washed twice with 500 µL of tween wash buffer, incubating at 55°C for 2 minutes with shaking for each wash. Beads were resuspended in 25 µL of Nextera Tagment DNA buffer. To tagment the captured DNA, 3.5 µL of Nextera Tagment DNA Enzyme 1 was added with 21.5 µL of Nextera Resuspension Buffer and samples were incubated at 55°C for 10 minutes with shaking. Beads were separated on a magnet and supernatant was discarded. Beads were washed with 500 µL of 50 mM EDTA at 50°C for 30 minutes, then washed three times with 500 µL of tween wash buffer at 55°C for 2 minutes each, and finally washed once with 500 µL of 10 mM Tris-HCl pH 7.5 for 1 minute at room temperature. Beads were separated on a magnet and supernatant was discarded. To generate the sequencing library, PCR amplification of the tagmented DNA was performed while the DNA is still bound to the beads. Beads were resuspended in 15 µL of Nextera PCR Master Mix, 5 µL of Nextera PCR Primer Cocktail, 5 µL of Nextera Index Primer 1, 5 µL of Nextera Index Primer 2, and 20 µL of water. DNA was amplified with 8 cycles of PCR. After PCR, beads were separated on a magnet and the supernatant containing the PCR amplified library was transferred to a new tube, purified using the Zymo DNA Clean and Concentrate-5 (Zymo D4003T) kit according to manufacturer's protocol, and eluted in 14 µL water. Purified HiChIP libraries were size selected to 300-700 bp using a Sage Science Pippin Prep instrument according to manufacturer's protocol and subject to paired-end sequencing on an Illumina HiSeq 2500. SMC1A libraries were initially sequenced with 100×100 bp paired-end sequencing.