GSM2348289: mESC_Hi-C 2; Mus musculus; OTHER
Between 25 to 50 x 10e7 cells were used to perform in-nucleus ligation 3C; biotin fill-in was omitted. Briefly, adherent cells (mES) were incubated in the presence of trypsin (Biological Industries 03-053-1) for 8-10 minutes max at 37°C and passed through a cell strainer after harvesting to ensure a single-cell suspension. Cells (including K562 from now on) were washed 1x with phosphate-buffered saline (PBS) and resuspended PBS/FBS 10% and cross-linked in formaldehyde final concentration of 2% for 5 minutes at room temperature. After quenching with 0.125 M glycine and wash with PBS, cells were incubated in permeabilization buffer (10 mM Tris–HCl pH 8, 10 mM NaCl, 0.2 % NP-40 alternative (CALBIOCHEM 492016), Protease Inhibitor Cocktail (P8340 Sigma)) for 60 minutes at 4°C while slowly mixing. Cells were split into 5 M cell aliquots and washed 2x with cold PBS. After the second wash, cell pellets were then flash frozen on liquid nitrogen. Pellets were resuspended on 175.5 µl H2O and 24.5 µl DpnII RE buffer 10x. After addition of 3 µl of 20% SDS (final concentration of 0.3%), cells were incubated for 60 minutes at 37°C while agitating (750 RPM). Triton X-100 20% was added to a final concentration of 1.8% and incubated for 60 min at 37°C. Aliquots were then digested with 15 µl HC DpnII (NEB 50,000 U/ml) ON. Cells were centrifuged for 5 minutes at 600 g and washed 1x with PBS. Incubation of pellets for 20 minutes at 65°C was performed in order to heat-inactivate any DpnII residue. In-nucleus ligation was then performed incubating cells on ligation mix (415 µl H2O, 5 µl BSA (10mg/ml NEB) and 5 µl T4 ligation enzyme NEB (2x106 U/ml)) ON at 16°C.  Cross-link was reversed by incubating aliquots with 10 mg/ml proteinase K (Roche) at 65°C ON followed by the addition of 15 µl of RNase A (Roche) for 45 minutes at 37°C. DNA was purified with AmpureXP SPRI beads 1x (Beckman Coulter; A63881) with two elution rounds of 100 µl each with the aim of size selecting for the high molecular weight portion of the sample. Hi-C: Bioruptor Microtubes (C30010015) to reach a DNA fragment size between 400-500 bp. The sonicated DNA  was then subjected to end-repair reaction (5 µl 10x end-repair buffer + 2.5 µl end-repair mix (NEB E6050L)) at 20 °C for 30 minutes. The DNA was cleaned up with 2.2x SPRI beads and eluted in 38 µl EB. A-tailing reaction was performed with 5 µl NEB buffer 2, 4 µl Klenow fragment -3′-5′ exo (NEB M0212M) and 10 µl dATP 10 nM for 30 minutes at 37 °C, cleaned up with 2.2 x SPRI beads and eluted in 30 µl EB. DNA template was ligated with 5 µl Illumina indexed adapters 0.75 µM with 5 µl T4 quick ligase (NEB M2200) and 40 µl 10x ligase buffer for 15 minutes at 25 °C. Ligated template was then cleaned up with 1.3x SPRI beads and eluted in 41 µl.