title: GSM3105137: Hi-C_HepG2; Homo sapiens; Hi-C
library_construction_protocol: Hi-C experiments have been conducted as previously described (Ramírez, Fidel, et al. "High-resolution TADs reveal DNA sequences underlying genome organization in flies." Nature communications 9.1 (2018): 189.), with the following modifications. Nuclei from cell pellets containing about 4 million of cells have been extracted by sonication (Arrigoni, Laura, et al. "Standardizing chromatin research: a simple and universal method for ChIP-seq." Nucleic acids research 44.7 (2015): e67-e67.) using the following parameters: 75W peak power, 2% duty factor, 200 cycles/burst, 180 seconds, using Covaris milliTubes and Covaris E220 sonicator. After nuclei permeabilization, chromatin has been digested overnight at $37ºC using HindIII high fidelity (80 units per million cells; R3104S, NEB). Biotin incorporation has been carried out at 37ºC for one hour in 300 μl volume using these reaction conditions: 50 mM of each nucleotide (dATP, dTTP, dGTP, biotin-14-dCTP, from Life Technologies, 19518-018), 8 U of Klenow (NEB, M0210L). Ligase mix has been added to each sample followed by 4 hours of incubation at room temperature under rotation. After nuclei lysis, protein digestion and overnight de-crosslink, DNA has been precipitated and sonicated to 100-600 bp. Biotinylated DNA has been pulled down as previously described. 100 ng of DNA bound to beads have been used for library preparation using a modification of the NEBNext Ultra DNA library preparation workflow (NEB, E7370). DNA bound to beads has been end-repaired, A-tailed, adaptor-ligated and USER-treated following manufacturer's instruction. After a bead wash, DNA has been eluted from the beads by incubating at 98°C for 10 minutes. Adaptor-ligated DNA has been PCR amplified using 7 PCR cycles. Libraries have been sequenced paired-end, with a read length of 75 bp, on the Illumina NextSeq 500 instrument.
attributes.tag: GEO Accession
attributes.value: GSM3105137