GSM4998182: 4Cseq_293T-WT_HIST2H2AB-bait_Rep2_R1; Homo sapiens; OTHER
For 4C-seq, HEK293T cells cultured in 10 cm dish were fixed with 4% formaldehyde for 10 min at room temperature. Cross-linked HEK293T cells were lysed, and the lysate was subjected to first restriction enzyme digestion using NlaIII (R0125S; NEB) followed by in situ ligation reaction using T4 DNA ligase (10799009001; Roche Diagnostics, Tokyo). After reverse-cross-linking, the ligated DNA templates were purified using P-beads (NucleoMag® NGS Clean-up and Size Select, 744970.5, Takara Bio Inc). Then, 2nd round of restriction enzyme digestion using DpnII (R0543S; NEB) and ligation was performed for trimming and circularization of template DNA. Resulting 4C-templates were purified by P-beads and subjected to sequencing library preparation. 4C-seq libraries werec generated essentially as described in Krijger et al., 2020 (Methods, 170: 17-32; doi: 10.1016/j.ymeth.2019.07.014). 4C-seq libralies were pooled and sequenced on Illumina Hiseq 2500 platform.