GSM9449570: H9-siNIPBL-NC_HiCP_CTCF_Brep7_Trep1; Homo sapiens; OTHER
HiChIP HiChIP libraries were generated using control (ctrl) and siRNA-treated H9 cells, along with their differentiated counterparts at various stages, as previously described, with some modifications. In brief, cells were collected and crosslinked with 1% formaldehyde for 10 minutes at room temperature, then quenched with glycine, washed with PBS, and stored frozen at -80°C. For library preparation, the fixed cells were gently homogenized using pestle A in Hi-C lysis buffer to release the nuclei, followed by digestion with DpnII, biotin-dCTP fill-in, and re-ligation. After ligation, chromatin was sheared by sonication into fragments of 200–500 bp and pre-cleared with Protein A and G Dynabeads at 4°C for 2 hours. Then, it was precipitated using Protein A or G Dynabeads that had been pre-incubated overnight with the appropriate antibodies to enrich the ligation products associated with specific proteins. Tagmentation of the immunoprecipitated chromatin was performed using a Tn5 transposase mixture on the beads. Following elution, reverse crosslinking, and ethanol precipitation, a second pull-down with streptavidin beads was conducted to enrich biotin-labeled ligation products. Finally, on-bead PCR amplification was performed to prepare the libraries for Illumina sequencing. Two biological replicates were created for each sample. 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.