GSM8173076: HiC_S7WT_rep1; Mus musculus; Hi-C
5 to10 million TTF cells were scrapped and resuspended in their culture medium supplemented with 10% FBS and 2% formaldehyde (Thermo Fisher #28908), followed by an incubation of 10 minutes rotating at room temperature to fixate the cells. The formaldehyde was quenched by adding cold glycine at a final concentration of 0.125M and incubating the cells for a further 5 minutes rotating at room temperature and 15 minutes on ice. The cells were washed twice with cold 1X PBS and nuclei were extracted by resuspending the cells in 1 ml of lysis buffer containing 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% IGEPAL CA630 (SIGMA-ALDRICH #18896-50ML), 1× cOmplete EDTA-free protease inhibitor cocktail (Merck #11873580001) and incubating the cells for 30 minutes on ice. Afterwards, the nuclei were washed with 1.25X NEBuffer 2 (NEB #B7002S) and resuspended in 358 μl of the same buffer. 11 μl of 10% SDS (AppliChem #A0676,0250) was added to remove proteins not crosslinked and the samples were incubated for 1 hour at 37ºC, followed by the addition of 75 μl of 10% Triton X-100 (AppliChem #A4975,0100) to quench the SDS and another 1 hour incubation at 37ºC. The chromatin was then digested overnight at 37ºC by adding 1500 U of HindIII (NEB #R0104T). The following day, the DNA ends were filled in with dCTP, dGTP, dTTP and biotin-14-dATP (Invitrogen #19524-016). In-nucleus ligation was then performed by adding 25 U of T4 DNA ligase (Thermo Fisher #15224025), 100 μl of 10× ligation buffer (NEB #B0202S), 10 μl of 10 mg/ml BSA (NEB # B9001S) and 170.5 μl of nuclease-free water and incubating the sample for 4 hours at 16ºC. 12.5 μl of RNase A was added and the nuclei were further incubated for 30 minutes at 37ºC. To reverse crosslinking, the samples were incubated overnight at 65ºC with 60 μl of 10 mg/ml Proteinase K (Roche #03115879001). The DNA was subsequently purified using a phenol : chloroform : isoamyl alcohol 25:24:1 extraction and ethanol precipitation. Non-informative biotins at the DNA ends were removed by incubating the samples with dATP and T4 DNA polymerase for 4 hours at 20ºC. The DNA was again purified using a phenol : chloroform : isoamyl alcohol 25:24:1 extraction and ethanol precipitation and quantified. 10 ug of DNA were sonicated using a Covaris M220 focused ultrasonicator in 130 μl cuvettes (70 s, Duty Factor 10%, Peak Incidence Power 50, Cycles per Burst 200). The sheared DNA ends were repaired by adding dNTPs, T4 DNA polymerase (NEB #M0203L), T4 PNK (NEB #M0201L) and Klenow enzyme (NEB #M0210L) and dATP-tailed by purifying the DNA using CleanNGS SPRI beads (CleanNA #CNGS-0050) and adding dATP and Klenow exo-enzyme (NEB #M0212L). The enrichment of informative DNA ligation products was carried out by pulling down those DNA fragments that contained a biotin using Dynabeads MyOne Streptavidin C1 paramagnetic beads (Thermo Fisher #65001) according to the manufacturer's instructions. The bead-bound DNA fragments were resuspended in 50 μl of 1x ligation buffer (NEB #B0202S) and the NGS library was constructed by adding 4 μl of 15 uM pre-annealed adapter mix (Illumina Truseq system, see supplementary Table 9 for sequences), 1μl of T4 DNA ligase (NEB #M0202T) and incubating the mix for 2 hours at room temperature. After washing the bead-bound DNA fragments with the adapters ligated, the library was amplified using illumine Truseq PCR primers (see Supplementary Table 9 for sequences) and Phusion high-fidelity PCR master mix with HF buffer (NEB #M0531L) for 8 cycles following the program: 98 °C for 40 s, followed by 8 cycles of 10 s at 98 °C, 30 s at 62 °C, and 30 s at 72 °C, and final extension for 10 min at 72 °C. Last, the library-containing supernatant was recovered from the beads, and the library size distribution was delimited between 300-800 bp using CleanNGS SPRI beads double-sided selection (0.4-1 volumes; CleanNA #CNGS-0050). Concentration and size distribution were assessed by automated electrophoresis (Agilent Tapestation platform with high sensitivity D1000 ScreenTape).