GSM4041376: mut CH12+FL-CTCF; Mus musculus; Hi-C
In situ Hi-C experiments were performed using the MboI restriction enzyme. The crosslinked pellets (1.5 million cells) were incubated and washed with 200 μL of lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA630, 33 μL Protease Inhibitor (Sigma, P8340)) on ice, and then incubated in 50 μL of 0.5% SDS for 10 min at 62°C. After heating, 170 μL of 1.47% Triton X-100 was added and incubated for 15 min at 37˚C. To digest chromatin 100 U MboI and 25 μL of 10X NEBuffer2 were added followed by overnight incubation at 37˚C. The digested ends were filled and labeled with biotin by adding 37.5 μL of 0.4 mM biotin-14-dATP (Life Tech), 1.5 μL of 10 mM dCTP, 10 mM dTTP, 10 mM dGTP, and 8 μL of 5 U/μL Klenow (New England Biolabs) and incubating at 23°C for 60 minutes with shaking at 500 rpm on a thermomixer. Then the samples were mixed with 1x T4 DNA ligase buffer (New England Biolabs), 0.83% Triton X-100, 0.1 mg/mL BSA, 2000 U T4 DNA Ligase (New England Biolabs, M0202), and incubated for at 23°C for 4 hours to ligate the ends. After the ligation reaction, samples were resuspended in 550 μL 10 mM Tris-HCl, pH 8.0. To reverse the crosslinks, 50 μL of 20 mg/mL Proteinase K (New England Biolabs) and 57 μL of 10% SDS were mixed with the samples, and incubated at 55°C for 30 minutes, and then 67 μL of 5 M NaCl were added followed by overnight incubation at 68°C. After cooling at room temperature, 0.8X Ampure (Beckman-Coulter) purification was performed, and the samples were sonicated to a mean fragment length of 400 bp using Covaris M220. Two rounds of Ampure (Beckman-Coulter) beads purification was performed for size selection. Biotin-labeled DNA was purified using Dynabeads MyOne T1 Streptavidin beads (Invitrogen). The beads were washed with 400 μL of 1x Tween Wash Buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20), and resuspended in 300 μL of 2x Binding Buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl). The beads were added to samples and incubated for 15 minutes at room temperature. Then the beads were washed twice by adding 600 μL of 1x Tween Wash Buffer. Then the beads were equilibrated once in 100 uL 1x NEB T4 DNA ligase buffer (New England Biolabs) followed by removal of the supernatant using a magnetic rack. To repair the fragmented ends, the beads were resuspended in 100 μL of the following: 88 μL 1X NEB T4 DNA ligase buffer (New England Biolabs, B0202), 2 μL of 25 mM dNTP mix, 5 μL of 10 U/μL T4 PNK (New England Biolabs), 4 μL of 3 U/μL NEB T4 DNA Polymerase (New England Biolabs), 1 μL of 5 U/μL Klenow (New England Biolabs). The beads were incubated for 30 minutes at room temperature. The beads were washed twice by adding 600 μL of 1x Tween Wash Buffer. To add a dA-tail, the beads were resuspended in 100 μL of the following: 90 μL of 1X NEBuffer2, 5 μL of 10 mM dATP, and 5 μL of 5 U/μL Klenow (exo-) (New England Biolabs). The beads were incubated for 30 minutes at 37˚C. The beads were washed twice by adding 600 μL of 1x Tween Wash Buffer. Following the washes, the beads were equilibrated once in 100 μL 1x NEB Quick Ligation Reaction Buffer (New England Biolabs) and the supernatants were removed using a magnetic rack. The beads were then resuspended in 50 μL 1x NEB Quick Ligation Reaction Buffer. To ligate adapters, 2 μL of NEB DNA Quick Ligase (New England Biolabs) and 3 μL of Illumina Indexed adapter were added to the beads and incubated for 15 minutes at room temperature. The supernatant was removed and the beads were washed twice by adding 600 μL of 1x Tween Wash Buffer. Then the beads were resuspended once in 100 μL 10 mM Tris-HCl, pH 8.0, followed by removal of the supernatant and resuspension again in 50 μL 10 mM Tris-HCl, pH 8.0. After deciding an optimal PCR cycle number using KAPA DNA Quantification kit (Kapa Biosystems), 6 cycles of PCR amplification were performed with the following reaction mixture: 10 μL Phusion HF Buffer (New England Biolabs), 3.125 μL 10 μM TruSeq Primer 1, 3.125 μL 10 μM TruSeq Primer 2, 1 μL 10 mM dNTPs, 0.5 μL Fusion HotStartII, 20.75 μL ddH20, 11.5 μL Bead-bound Hi-C library. PCR products were subjected to a final purification using AMPure beads (Beckman-Coulter) and were eluted in 30 μL 10 mM Tris-HCl, pH 8.0. Libraries were sequenced on the Illumina HiSeq 4000 platform.