title: GSM2978427: HiC_DMSO_4h; Homo sapiens; Hi-C
library_construction_protocol: For HiC experiments, 5-10 million purified CD4+ T lymphocytes were fixed for 10 min with 1 % formaldehyde after 4h treatment with 20 mM UNC4976 (0.2% DMSO) or 0.2% DMSO. The fixing was quenched by adding glycine (100 mM final concentration). After pelleting at 1200g and decanting, cells were resuspended with 10 mL 3e Hi-C lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% NP40, and Sigma protease inhibitor cocktail) and incubated at 4 °C for 1 h. The cells were pelleted and resuspended with 400 mL CutSmart Buffer (1X, NEB), adjusted to 0.1% SDS, and heated at 65 °C for 10 min. The mixture was treated with 10 % Triton X-100 (44 mL), and digested with CviQI (20U, NEB) and CviAII (20U, NEB) at room temperature for 20 min. Bfa I was added (20U, NEB) and the mixture was incubated at 37 °C for 20 min. The reaction mixture was quenched with the addition of 600 mL wash buffer (10mM NaCl, 1mM EDTA, 0.1% Triton X-100). The cells were pelleted, and washed again with 600 mL wash buffer. Blunt-ended DNA fragments were generated by incubating with Klenow Fragment (NEB) for ligation of dCTP, dGTP, dTTP, biotin-dATP, followed by blunt-end ligation with T4 DNA ligase (NEB) at 16 °C overnight. The samples were de-crosslinked, treated with T4 DNA Polymerase, and sheared to a median fragment length of 400 bp with Diagenode sonicator. After DNA End Repair and A-tailing according to standard procedure31 and biotin capture was performed with Streptavidin C1 beads (ThermoFisher) according to manufacturer's specifications. On-bead ligation was carried out with Paired-End Adaptors (Illumina) and PCR amplification and size selection afforded indexed sequencing libraries with a median fragment length of 500 bp. After 2% E-gel purification (ThermoFisher), libraries were paired-end sequenced on the HiSeq 2500.
attributes.tag: GEO Accession
attributes.value: GSM2978427