title: HiC of human: LHCN-M2 Cells-WT1
design: The cells were cross-linked with 1% formaldehyde for exactly 10 min at room temperature. Add 1.25 ml of 2.5 M glycine to quench the cross-linking reaction for 5 min at room temperature, and then place on ice for at least 15 min. pellet the cell with 1000g for 10 min. Wash the pellet with DPBS, then add were cross-linked with DSG (final concentration 3mM) for 40 min at room temperature.  Cross-linked cells were lysed in ice-cold lysis buffer containing 10 L protease inhibitor cocktail. and the nuclei were digested with 400 U DdeI and 400 U DpnII (NEB) at 37 C overnight with interval shaking (900 rpm, 30 s on, 4 min off). Incubate the Hi-C samples with Biotinylation buffer(7 L 10 NEBuffer 3.1, 1.5 L 10 mM dCTP, 1.5 L 10 mM dGTP, 1.5 L 10 mM dTTP,  37.5 L 0.4 mM biotin-14-dATP, 11 L H2O, 10uL Klenow DNA polymerase) 4 hours at 23C in a Thermomixer (900 rpm, 30 s on, 4 min off).  Incubate ligation Mix ( 240 L 5 ligation buffer, 120 L 10% Triton X-100, 12 L 10 mg/ml BSA, 243 L H2O, 50 L 1 U/ L T4 DNA ligase) with biotinylation mix for 4 hr at 16C in a Thermomixer with interval shaking(900 rpm, 30 s on, 4 min off). To each Hi-C sample, add 50 L of 10 mg/ml Proteinase K and incubate 2 hours at 65C with interval shaking (900 rpm, 30 s on, 4 min off), then another 50 l of 10 mg/ml Proteinase K is add to each Hi-C sample and continue incubating overnight at 65C. Prepare biotin removal reactions in PCR tubes as described below: 15 g of Hi-C DNA sample, 13 L 10 NEB buffer 2.1, 3.25 L 1 mM dATP,  3.25 L 1 mM dGTP, 13 L 1 mM 3000 U/ml T4 DNA polymerase, up to 130 L with water. Transfer two 65-l aliquots from each 130-l reaction to a thermocycler with following cycling parameters: 20 C for 4 hr, 75C for 20 min, hold at 4C. Sonicate samples on Covaris E220 Evo sonicator to shear a fragment size distribution 300-500bp. Double size selection was performed on sheared DNA using 0.8X-1.1X  Agencourt AMPure XP beads (A63881, Beckman Coulter) according to -manufacturers instructions. Incubate end-repair mix (7 L 10 ligation buffer, 7 L 2.5 mM dNTP mix, 2.5 L 3 U/L T4 DNA polymerase, 2.5 L 10 U/ L T4 polynucleotide kinase, 0.5 L 5 U/ L Klenow DNA polymerase, H2O 4.5 L) and size-selected HiC libraries in a thermocycler with following cycling parameters:  20C for 30 min, 75C for 20 min, hold at 4C. The products were then purified with MyOne Streptavidin C1 beads (65001, Thermo Fisher Scientific), dA-tailed, ligated to 3 mL 15 mM NEBNext Adaptor for Illumina at 37 C for 2h.  Hi-C libraries were then amplified with 4 cycles of PCR. In situ Hi-C on WT and Smchd1-/- LHCN-M2 cells were performed in two biological replicates, which were sequenced on Illumina HiSeq with 150-bp paired-end read runs