GSM7405563: HEK293T_1000cells_2; Homo sapiens; Hi-C
To gather the cells for Hi-C processing, the cells were washed twice using PBS, detached by adding 1 mL 0.25% trypsin-EDTA to their culture dish, centrifuged at 500g for 5 min, and recovered in PBS buffer. The cells were counted by a cell counter to determine the concentration.For sample 0923-4, 0716HEK293T-1, 0716HEK293T-2, 0716HEK293T-3 and 0716HEK293T-4, 1000 cells were extracted to a 0.5 mL Eppendorf Lobind Microcentrifuge tube (Eppendorf, #32119210) for each sample. For sample 1002-5 and 0923-2, 10,000 cells were extracted. The libraries were consturcted following the Hi-C method described in Rao et al., 2014 (PMID: 25497547) with some modification. Briefly, the fixed cell pallet was lysed in 100 μL Hi-C lysis buffer on ice for at least 30 min. Permeabilision was performed by adding 50 μL of 0.5% SDS then quenched in 25 μL of 10% Triton X-100. Chromatin was digested by MboI enzyme. After digestion, fill-in mix was added to mark DNA ends. T4 DNA ligase was used to carry out the proximity ligation reaction. DNA fragments were released by Proteinase K. The DNA fragments were purified by Ampure XP beads and Tagemtation was performed using Tn5 enzyme. Dynabeads M-280 streptavidin was used to perform biotin pull-down. PCR amplification was carried out to obtain sufficient library fragments for the sequencing.