title: GSM4996035: ESC_LMNA_ctr_HIC_seq_Rep3; Mus musculus; Hi-C
library_construction_protocol: Hi-C libraries were generated according to (Rao et al., 2014) with modifications. Five million mESC were crosslinked with 1% formaldehyde for 10 min at RT and quenched with 120µM Gylcine for 5 min at RT on a rotator. Cells were pelleted at 4 °C for 5 min at 2,500 relative centrifugal force (RCF) and washed twice with ice cold PBS. Cells were lysed om 500 μL of ice-cold Hi-C lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2% NP-40, 1× protease inhibitors) with rotation at 4 °C for 30 min. Nuclei were then pelleted and resuspended in 100 μL of 0.5% SDS and incubated at 62 °C for 10 min. The SDS reaction were stopped by adding 285 μL of water and 50 μL of 10% Triton X-100 to each sample at 37 °C for 15 min. 200 U of MboI restriction enzyme (NEB, R0147) and 50µl of NEB Buffer 2 were used to digest the chromatin. Samples were then incubated at 37 °C for 3 h with rotation. The restriction enzyme was then heat inactivated at 62 °C for 20 min. To fill-in the MboI overhang and mark the ends, 52 μL of incorporation master mix ( 37.5 μL of 0.4 mM biotin–dATP (Thermo Fisher, 19524016); 4.5 μL of a dCTP, dGTP, and dTTP mix at 10 mM each; and 10 μL of 5 U/μL DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210)) were added to the nuclei and incubated at 37 °C  for 1 h with rotation. 948 μL of ligation master mix (150 μL of 10× NEB T4 DNA ligase buffer with 10 mM ATP (NEB, B0202), 125 μL of 10% Triton X-100, 3 μL of 50 mg/mL BSA (Thermo Fisher, AM2616), 10 μL of 400 U/μL T4 DNA Ligase (NEB, M0202), and 660 μL of water ) were then added to each samples and incubated overnight at 16 °C. After proximity ligation, the nuclei were pelleted at 2,500 RCF for 5 min at room temperature and the supernatant was used further. Reverse crosslinking were done by overnight incubation at 65°C. DNA was purified using ChIP DNA Clean & Concentrator Kit (Zymo Researhc, D5205) and then sheared to a length of ∼400 bp. 50 ng of the sheared DNA was used for pull down of biotinylated proteins with 5 μL of Streptavidin C-1 beads (Thermo Fisher, Cat. N) in biotin binding buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl) at room temperature for 15 min while shaking. Beads were then captured on the magnet, washed twice with 500 μL of Tween Wash Buffer at 55 °C for 2 min with shaking followed by one washing step using 100 μL of 1× TD Buffer (10 mM Tris-HCl pH 7.5, 5 mM magnesium chloride, 10% dimethylformamide). The beads were then resuspended in 25 μL of 2× TD Buffer, 2.5 μL Tn5 and water to 50 μL and incubated at 55 °C for 10 min while shaking. The beads were captured with a magnet and incubated in 50 mM EDTA at 50 °C for 30 min while shaking. Beads were further washed two times with 50 mM EDTA followed by a wash with Tween Wash Buffer at 55 °C for 2 min and with 10 mM Tris before PCR amplification. Beads were then resuspended in 25 μL of Phusion HF 2× (New England Biosciences), 1.25 μL of illumine Index 1 (i7) adapters and Index 2 (i5) Adapters (from Nextera kit, Cat N) and 22.5 μL of water and amplification was carried out using the following PCR program: 72 °C for 5 min, 98 °C for 1 min, then cycle at 98 °C for 15 s, 63 °C for 30 s, and 72 °C for 1 min. Cycle numbers needed for amplification (beginning of exponential amplification) was determined for each sample. Afterwards, Ampure beads were used for double size selection to purify DNA with size range of 300- 700 bp. After size selection, libraries were quantified on Bioanalyzer and sequenced paired end with read length of 75.