GSM9024514: Hepatocyte-like cells, Etoposide, Replicate2; Homo sapiens; Hi-C
Nuclei was extracted from samples. The nuclei pellet was resuspended in the Tn5 transposase reaction mix. The transposition reaction was incubated at 37°C for 30 min. Equimolar Adapter1 and Adatper 2 were added after transposition, PCR was then performed to amplify the library. After the PCR reaction, libraries were purified with the AMPure beads and library quality was assessed with Qubit. Hi-C library preparation was performed according to the Hi-C 2.0 protocol with minor modifications. Briefly, 1–5 million cells were incubated in trypsin for 15 minutes to facilitate detachment, then pelleted by centrifugation at 500g, washed twice with PBS, and fixed in 1% formaldehyde for 10–12 minutes. Fixation was quenched with glycine, after which cells were pelleted, washed, and stored at –80°C. For library preparation, frozen cells were thawed on ice, lysed, and digested overnight with 300 U of DpnII. The resulting DNA ends were labeled with biotin-14-dCTP using Klenow fragment and ligated overnight with T4 DNA ligase. Crosslinks were reversed by Proteinase K treatment at 65°C overnight, followed by ethanol precipitation. Unligated biotinylated ends were removed using T4 DNA polymerase.Next-generation sequencing (NGS) libraries were prepared using the KAPA HyperPlus Kit. Biotin-labeled ligation products were selectively enriched using Dynabeads MyOne Streptavidin C1 beads prior to library amplification. Sequencing was performed using 150 bp paired-end reads.