GSM8394769: 44C HiC biol rep 2; Escherichia coli; Hi-C
Cell fixation, lysis and restriction enzyme digestion were performed as described above for Red-C with the only difference that 10 µL HpaII (50 U/µL, NEB) was used instead of NlaIII and the digestion was carried out for 3 h instead of 2 h. After HpaII digestion, the sample was pelleted for 10 min at 21000 g and 10 °C and resuspended in 250 µL 1× NEB buffer 2 followed by adding 42 µL fill-in solution composed of 15 µL 1 mM Biotin-dCTP, 1.5 µL 10 mM dAGTTP (mix of dATP, dGTP, and dTTP at 10 mM each), and 25.5 µL water. The mixture was supplemented with 8 μL Klenow (5 U/μL, NEB) and incubated for 2 h at 25 °C with shaking at 900 rpm. The sample was pelleted as above and resuspended in 300 µL 1× T4 DNA ligase buffer (ThermoFisher) followed by adding 12.5 µL T4 DNA ligase (5 Weiss U/µL, ThermoFisher). DNA proximity ligation was carried out overnight at 20 °C with shaking at 1000 rpm. The sample was pelleted as above and resuspended in 375 µL Proteinase K buffer composed of 4 µL 1 M Tris pH 8.0, 8 µL 5 M NaCl, 1.6 µL 0.5 M EDTA, 40 µL 10% SDS, and 321 µL water followed by adding 25 µL proteinase K (20 mg/mL, Ambion) and incubating for 8 h at 65 °C. Downstream steps of the Hi-C procedure including DNA purification, sonication, biotin-pull down, library construction, sequencing and read processing were done as described above for Micro-C.