GSM6543674: HiC_shScr; Mus musculus; Hi-C
In situ Hi-C: As described in Rao et al. (2014), 5 million cells were crosslinked with 1% formaldehyde for 10min. Crosslinked cell pellets were lysed with 300uL ice-cold HiC lysis buffer (10mM Tris HCl pH 8, 10mM NaCl, 0.2% Igepal CA630) with protease inhibitors (Roche). Cell suspension was incubated for 30min at 4C with rotation, then pelleted at 2500xg for 5min. Pelleted nuclei were then washed with 500ul ice-cold HiC lysis buffer and resuspended in 50uL 0.5% SDS solution and incubated at 62C for 10min. 145ul H2O and 25ul 10% Triton X-100 were added to quench SDS and nuclei incubated at 37C for 15min. 26.1ul of 10X NEB Buffer 2 and 100U of MboI restriction enzyme were added and nuclei digested overnight at 37C with rotation. MboI was then heat activated at 62C for 20min, then nuclei cooled to room temperature. To fill in MboI fragment overhangs and mark DNA ends with biotin, 50ul of fill-in master mix was added (37.5ul 0.4mM biotin-14-dATP, 1.5ul 10mM dCTP, 1.5ul 10mM dGTP, 1.5ul 10mM dTTP, 8ul 5U/ul DNA Polymerase I, Large (Klenow) Fragment) and mixed by pipetting. Nuclei were then incubated at 37C for 1h with rotation. To ligate biotinylated DNA ends together, 900ul ligation master mix was added (649ul H2O, 120ul 10X NEB T4 ligase buffer, 100ul 10% Triton X-100, 6ul of 20mg/ml BSA, 25ul of 400U/ul T4 DNA ligase). Reaction was mixed by inverting and incubated at room temperature for 4h with rotation. Reaction was then centrifuged at 1700xg for 5min at room temperature. Pelleted nuclei were resuspended in 750ul TES buffer (1mM EDTA, 10mM Tris-HCl pH 8, 0.5% SDS), 50ul Proteinase K (20mg/ml) added and incubated at 55C for 1h with rotation. To reverse crosslinks, 88.9ul 3M NaCl was added and crosslinks reversed by incubated overnight at 65C with shaking (Eppendorf thermomixer, 950rpm). Samples were cooled to room temperature and DNA extracted with phase-lock gel tubes (5Prime). RNA was digested from purifying DNA by adding 2ul RNAse A (20mg/ml) and incubating at 37C for 1h. DNA was then extracted with phase-lock gel tubes (5Prime) and resuspended in 130ul 1X Tris buffer (10mM Tris-HCl, pH 8). To ensure complete resuspension, DNA was then incubated at 37C for 15min. DNA was then sonicated to a size of 300-500bp using a Covaris with the following parameters: 140W, 10% duty cycle, 200 bursts/cycle, 100s. Samples were then size selected to 300-500bp using AMPure XP beads and eluted in 300ul 1X Tris buffer. To pulldown biotinylated ligation products, 30ul (per sample) of MyOne Streptavidin C1 beads were washed with 400ul of 1X Tween washing buffer (5mM Tris-HCl pH 7.5, 0.5mM EDTA, 1M NaCl, 0.05% Tween 20) and resuspended in 300ul 2X binding buffer (10mM Tris-HCl pH 7.5, 1mM EDTA, 2M NaCl). Beads were then added to purified DNA and sample incubated at room temperature for 15min with rotation. Beads were washed twice with 600ul 1X Tween Washing buffer. Subsequent library preparation steps were then done directly on beads. 30ul of Dynabeads MyOne Streptavidin C1 beads was used per sample. After adaptor-ligation with 3 ml of 15uM  NEBNext adaptor for Illumina (included in NEBNext Multiplex Oligos for Illumina; NEB, E7335S) at RT for 15 min, the C1 beads were separated on a magnet, washed with 600 ml of 1X TWB (5mM Tris-HCl, pH 7.5, 0.5mM EDTA, 1M NaCl, 0.05% Tween 20) twice at 55 C for 2 min and washed briefly with 100 ml of 1X NEBuffer 2.1 at RT. The resulting C1 beads were resuspended in 50 ml of 1XNEBuffer 2.1 with 3 ml USER enzyme (included in NEBNext Multiplex Oligos for Illumina; NEB, E7335S) and incubated at 37C for 20 min with rotation. After the buffer was discarded, the beads were resuspended in 100 ml of 1X PCR master mix [50 ml of Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531S), 2.5 ml of universal primer (NEBNext Multiplex Oligos for Illumina; NEB,E7335S), 2.5 ml of index primer (NEBNext Multiplex Oligos for Illumina; NEB, E7335S, 45 ml of water]. In situ Hi-C libraries were directly amplified from the beads with 12 cycles. Libraries were generated in two biological replicates.