GSM8625493: HiC, H9 cell line, with VEGF-A 12hr, rep1; Homo sapiens; Hi-C
Cells were harvested and crosslinked in 1% formaldehyde for 10 min at room temperature. Crosslinked cells were lysed with 10nM Tris-HCl (pH8.0), 10mM NaCl, and 0.2% IGEPAL CA630 (Sigma-Aldrich, 18896), and digested with 100U MboI (NEB, R0147). Digested fragments were labelled with biotin-14-dCTP (Invitrogen, 19518018) and proximally ligated with T4 DNA Ligase (NEB, M0202), followed by reverse-crosslinking with 2 ug/uL proteinase K (NEB, P8102), 1% SDS, and 500mM NaCl overnight at 68°C. The DNA fragments were purified with AMPure XP beads (Beckman Coulter, A63880), and subjected to sonication (Covaris, S220). The ligated DNA fragments were pulled down with Dynabeads MyOne streptavidin T1 beads (Invitrogen, 65602) with thorough washing. Hi-C libraries were prepared manually by performing DNA end repair, removal of un-ligated ends, adenosine addition at 3' end (NEB, M0212), ligation of Illumina indexed adapters (NEB, M2200), and PCR amplification (Thermo Fisher Scientific, F549). The number of cycles for PCR amplification was determined based on KAPA library quantification kit (KAPA, KK4854).