GSM4633885: dm_RA_d4; Mus musculus; Hi-C
Cells were crosslinked for 10 mins at room temperature in 1% methanol-free formaldehyde at a density of 106 cells/ml, quenched for 5 mins with 125mM glycine at room temperature. Cells were split 1.5 107 crosslinked cells were resuspended in 500 μL Hi-C Lysis Buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% NP40, 1X protease inhibitors) for 30 mins at 4°C on a rotating wheel, then centrifuged 5 mins at 2500g, and subsequently washed with 500 μL Hi-C Lysis Buffer and resuspended in 100 μL 0.5% SDS. Cells were then incubated  at 62°C for 10 minutes, after which 285 μL of H2O and 50 μL of 10% Triton X-100 were added to quench the SDS, and incubated at 37°C for 15 mins.  50 μL 10X NEB Buffer 2 and 375 U of MboI restriction enzyme (NEB, R0147) were added to digest chromatin for 2 hours at 37°C with rotation, followed by MboI inactivation at 62°C for 10 minutes. Restriction fragment overhang filling and biotin marking of DNA-ends was carried out in 52 μL Master Mix (0.4 mM biotin-dATP (Thermo 19524016), 10 mM dCTP/dGTP/dTTP, 5U/μL DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210)), mixed and incubated at 37 for 1 hour with rotation. 948 μL ligation master mix was subsequently added  (10X NEB T4 DNA ligase buffer with 10 mM ATP (NEB, B0202), 10% Triton X-100, 50 mg/mL BSA, 400 U/μL T4 DNA Ligase (NEB, M0202)), incubated at room temperature for 4 hours with rotation. Nuclei were then centrifuged at 2500g for 5 mins .For sonication, pellets were resupspended in 880 μL in Nuclear Lysis Buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS, 1X protease inhibitors), and sonicated in a Covaris militube using a Covaris E220 sonicator (fill level: 10, duty cycle: 5, PIP: 140, cycles/burst: 200, time: 4 mins). Biotin pull-down was carried out with 5 μL Streptavidin C-1 beads washed with Tween Wash Buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1M NaCl, 0.05% Tween-20) in 10 μL 2X Biotin Binding Buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl). Beads were incubated at room temperature with 50ng chromatin for 15 minutes with rotation, magnet-separated and washed twice with 500 μL Tween Wash Buffer at 55 °C for 2 minutes with shaking and subsequently washed in 100 μL of 1X TD Buffer (10 mM Tris-HCl pH 7.5, 5 mM MgCl2, 10% dimethylformamide), and resuspended in 25 μL 2X TD Buffer, to which 2.5 μL Tn5 transposase was added, and subsequently incubated at 55 °C with interval shaking for 10 minutes. Beads were magnet-separated and incubated in 50 mM EDTA at 50°C for 30 mins, washed twice in 50 mM EDTA at 50°C for 3 mins, and twice in Tween Wash Buffer at 55°C for 2 minutes, as well as once in 10 mM Tris For PCR and Post-PCR Size Selection, beads were resuspended in 50 μL of PCR master mix (Phusion HF 2X, Nextera Ad1.1 (Universal) 12.5 uM, Nextera Ad2.x (Barcoded) 12.5 uM), with 15 cycles (98°C 15 s, 63°C 30 s, 72°C 1 min). Libraries were cleaned up with 1.8X and subsequently 0.6X Ampure XP beads, and finally eluted in 10 μL water. Sequencing was carried out on an Illumina HiSeq 2500 sequencer.