GSM7851485: mAID-Mcm6 Control Asynchronous ESCs Replicate 1, Hi-C; Mus musculus; Hi-C
Hi-C experiments were performed as previously reported with some modifications (Kadota et al., 2020). Cells were fixed with 1% formaldehyde for 10 min at room temperature. Glycine was added to 0.125 M final concentration and incubated for 5 min at room temperature. Tubes were placed on ice for 15 min. Cells were washed in ice-cold PBS by centrifuging at 400 g at 4°C. Then, 1–2 x 10^6 fixed cell pellets were used for the following steps. Cell pellets were resuspended in 400 µl of lysis buffer (10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 0.2% NP-40) with 1x protease inhibitor cocktail (Roche, 11873580001), transferred to 1.5 ml Protein LoBind Tubes (Eppendorf) and incubated on ice for 20 min. The samples were spun down for 5 min at 1000 g at 4℃. After removing the supernatants carefully, pellets were resuspended in 250 µl of 1x NEBuffer 2 with 0.3% SDS and incubated for 1 h at 37°C in the Thermomixer C3 at 1100 rpm (Eppendorf). Then, 28 µl of 20% Triton X-100 were added to 2% final concentration and the samples were incubated for 1 h at 37°C. Next, 100 U of DpnIII (NEB, R0543S) was added and the samples were incubated overnight at 37°C in the Thermomixer C3 at 1100 rpm. The next day, after the inactivation of enzyme activity with 65˚C for 20 min, the samples were spun down for 5 min at 4°C. After removing the supernatants carefully, pellets were resuspended in 100 µl of “Biotin fill-in mix” (1 x NEBuffer 2, 15 µM each of dCTP, dGTP, dTTP and 15 µM of Biotin-14-dATP (Invitrogen, 19524-016), 15 U of Klenow (NEB, M0210S), 1% Triton X-100) and incubated for 45 min at 25°C. After spinning down for 5 min at 4°C and removing the supernatants carefully, the pellets were resuspended in 100 µl of “Ligation Mix” (1x T4 DNA ligase buffer (NEB), 6 µl of T4 DNA ligase (1 U/µl, Invitrogen, 15224-025), 1% Triton X-100) and incubated 6 hours at 16°C. The sample volumes were brought up to ~100 µl by adding 10 mM Tris-HCl (pH 8.0) / 5 mM EDTA and RNaseA-treated for 30 min at 37°C by adding 5 µl of 10 mg/ml RNaseA. Reverse-crosslinking and proteinase K treatment were performed overnight at 65°C by adding "Reverse-crosslink Mix" (1.5% SDS, 375 mM NaCl, 1 mg/ml Proteinase K). The next day, 5 µl of 20 mg/ml Proteinase K was added and incubated for 2 h at 55°C. Samples were then extracted twice with phenol/chloroform and once with chloroform. After this, 500 µl of 2-propanol was added. The samples were incubated for 10 min and spun down for 30 min at 20,000 g at 4°C for DNA precipitation. After 70% EtOH washes (add 1 ml, cfg for 5 min at 20,000 g at 4°C), the DNA pellets were resuspended in 30 µl of EB (QIAGEN, 19086). For generating next-generation sequencing libraries, 1-2 µg of Hi-C DNA was used per library. To remove biotin from unligated ends, Hi-C DNA was mixed with 50 µl of “T4 DNA pol mix” (1x NEBuffer 2.1, 100 µM dATP, 100 µM dGTP, 2.5 U of T4 DNA polymerase (NEB, M0203S)) and incubated for 30 min at 37°C. The sample volumes were brought up to ~130 µl by adding TE buffer. DNA samples were sheared (300–500 bp) using the following settings in Covaris S220 (Duty Factor: 5.0, Peak incident power: 175 W, Cycles per burst: 200, Treatment time: 120 sec) using microTUBE with snap cap (Covaris, 520045). After DNA shearing, DNA size selection was performed using AMPure XP beads (Beckman Coulter). Using the size-selected DNA samples, biotin pull-down and sequencing library preparations were performed using 30 µl of Dynabeads M-280 Streptavidin (Invitrogen, 11206D) and a part of KAPA LTP kit (KAPA BIOSYSTEMS, KK8232) following the kit's protocol. For library amplification, KAPA Real-Time Amplification Kits (KAPA BIOSYSTEMS, KK2701) were used to determine the optimal PCR cycles (5–8 cycles) and the final PCR products were purified by AMPure XP beads.