GSM9067548: Th1 cells, CBS-70, CTCF-binding site-70 deletion; Mus musculus; Hi-C
Cells were crosslinked with 3 mM disuccinimidyl glutarate and 1% formaldehyde. Intact nuclei were extracted with Micro-C Buffer #1 and digested with 20 U MNase for 10 minutes. Digested chromatin was washed twice with MB#2 and incubated in end-repair reactions for 15 minutes. To generate 5' fragment overhangs for end-blunting and labeling, 50 U Klenow Fragment was added and incubated for 15 minutes. A mixture of dNTPs in end-labeling buffer was added and incubated for 45 minutes. Samples were washed with MB#3 and proximity ligation was performed in a ligation reaction. To remove biotinylated dNTPs, samples were digested by Exonuclease III for 15 minutes. To prepare ligated DNA for library preparation, dinucleosome-sized DNA fragments (250 to 400 bp) were gel-extracted and purified with Dynabeads MyOne Streptavidin C1. Illumina library preparation was performed using the NEBNext Ultra II kit (NEB) and NEB Multiplex Oligos for Illumina. 120-mer probes were designed to tile end-to-end across the capture locus (mm10, chr10:118,369,795-118,508,916), and capture enrichment was performed according to the xGen Hybrid Capture protocol (Integrated DNA Technologies). Pooled libraries were sequenced with 150 bp paired-end reads using NovaSeq (Illumina).