GSM8773967: H9-siNIPBL-TPA200uM2hXNC_HiC_Brep2_Trep1; Homo sapiens; Hi-C
In-situ Hi-C In-situ Hi-C libraries were generated using the DpnII restriction enzyme, following previously established protocols. In summary, 2.5 million cells from each differentiation stage were crosslinked using 1% formaldehyde, quenched with glycine, washed with PBS, and permeabilized to isolate intact nuclei. The nuclear DNA was then digested with DpnII, with 5'-overhangs filled in with biotinylated dCTPs and dA/dT/dGTPs to create blunt-end fragments. These fragments were subsequently ligated, reverse-crosslinked, and purified using standard DNA ethanol precipitation methods. The purified DNA was sonicated to generate fragments of 200-500 bp in size and captured using streptavidin beads. The standard Illumina TruSeq library preparation protocol was followed, which included end-repair, A-tailing, and ligation with universal adapters, with the beads being washed twice in Tween Washing Buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween 20) between each step. The DNA attached to the beads was PCR amplified using barcoded primers and KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems) for 5 to 12 cycles to ensure adequate DNA quantity for sequencing. The libraries were then paired-end sequenced on an Illumina NovaSeq 6000 platform. More than two biological replicates were created, and the replicates were combined for all analyses after confirming a high level of correlation. Truseq libraries were constructed using the standard protocol. Genomic fragments were end repaired (NEBNext End Repair Module), A-tailed by adding adenosine to the 3' ends of fragment using Klenow fragment (3' to 5' exo minus, New England Biolabs), and adaptors were ligated at room temperature for 1 hr with T4 DNA ligase (New England Biolabs). Libraries were amplified with Illumina primers using the KAPA SYBR FAST qPCR Master Mix. Nextera libraries were amplified with Nextera barcoded primers using the KAPA SYBR FAST qPCR Master Mix. Illumina libraries were sequenced paired-end at 50 bp length on Illumina Hiseq2500  v4 or NovaSeq  6000  instruments.