title: GSM9083507: Trim66_KO_het_HiC_rep_1; Mus musculus; Hi-C
library_name: GSM9083507
library_construction_protocol: The live cells were fixed with 1% formaldehyde for 10 min and quenched with 0.2 M glycine for 5 min at room temperature. The cells were then washed twice with 100 uL HBSS containing 0.1% BSA. The pelleted cells were resuspended in 100 uL of ice-cold Hi-C lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630, and protease inhibitors) and incubated on ice for 30 min. After centrifugation at 3,000 g for 5 min at 4 °C, the pelleted nuclei were washed once with 100 uL of ice-cold 1x NEB Buffer 3.1. Chromatin was solubilized in 20 uL of 0.3% SDS and incubated at 65 °C for 10 min, followed by quenching with 5 uL of 10% Triton X-100 and incubation at 37 °C for 10 min. The nuclei were digested with 50 U DpnII in a total volume of 50 uL at 37 °C for 3 h in a Thermomixer with interval shaking. DpnII was then inactivated by incubation at 65 °C for 20 min. For biotin labeling, 3 uL of 1 mM biotin-14-dATP, 0.3 uL of 10 mM dGTP, 0.3 uL of 10 mM dTTP, 0.3 uL of 10 mM dCTP, and 10 U Klenow were added. The reaction was performed at 37 °C for 1.5 h in a Thermomixer. Subsequently, 60 uL of ligation mixture (33.6 uL water, 12 uL 10x NEB T4 DNA ligase buffer, 12 uL 10% Triton X-100, 1.2 uL 10 mg/mL BSA, and 1.2 uL 2,000 U/uL T4 DNA ligase) was added, and the reaction was incubated at 25 °C for 4 h in a Thermomixer. Crosslinks were reversed and DNA was purified. The purified DNA was sheared to 300-500 bp using a Covaris M220. End repair, dA-tailing, and adaptor ligation were then performed using the Rapid Plus DNA Lib Prep Kit V2 (ABclonal). Biotin-labeled DNA fragments were enriched using 50 uL of Dynabeads MyOne Streptavidin C1 (Invitrogen). Sequencing libraries were prepared on beads, followed by 6-8 cycles of PCR amplification. Fragments ranging from 300 bp to 1,000 bp were size-selected. All libraries were sequenced on a NovaSeq X Plus platform following the manufacturer's instructions.
identifiers.namespace: GEO
identifiers.id: GSM9083507_r1