GSM4120560: hic_minus35.inter_30_10000_blocks; Homo sapiens; Hi-C
Suspension cells (SEM) cells were collected at 1X107 cells per reaction while adherent cells (IMR90, HCT116 and human iPS) were trypsinized before collection. Cells were fixed and lysed in 15 mL conical tubes before storage in -80°C for up to 1 month before library construction. Cells were thawed on ice and spun down for 5 minutes at 4000rpm, 4°C. The remaining lysis buffer was removed and discarded. The pellets were re-suspended in 1 mL of 1x DpnII buffer and transferred to a 2 mL tube for homogenization with a pestle. The homogenized samples were then centrifuged for 14,000 rpm at 5 mins in 4°C. The supernatant was removed and discarded. After decrosslinking, phenol-chloroform-isoamylalcohol DNA extraction, and sodium acetate precipitation, a small portion of DNA was run on a gel to check for digest and ligation efficiency. Once the 3C library was construction was complete, 5-6ug of the 3C DNA in a total volume of 130ug per covaris tube (microTUBE AFA Fiber Crimp-Cap 6X16mm, catalog). Each 2ug of 3C library generated one Capture.NEBNext Ultra II DNA library kit for Illumina (NEB 7645S) was used for end-repair, adaptor ligation, and PCR enrichment of adaptor ligated DNA. NEBNext Mulitplex Oligos (Dual Index Primers Set 1) was used for indexing for four cycles.The Nimblegen SeqCap EZ Hybridization and Wash Buffer Kit (Roche) was used for subsequent hybridization and wash steps. Constructed library was sequenced on Miseq for paired-end 150bp. Briefly, five million CTCFAID SEM cells (clones 27 and 35) with or without IAA treatment were crosslinked with 1% formaldehyde for 10 min at room temperature, digested with 125 Units of MboI, labeled by biotinylated nucleotides and ligated for proximity ligation. After de-crosslinking, ligated DNA was purified and sheared to 300–500 bps. Ligation junctions were then pulled down with streptavidin beads and prepped as a standard Illumina library. Each library underwent 75-cycle paired-end sequencing on Illumina HiSeq 4000 system. About 100 million reads were obtained for each sample.
GEO Accession
GSM4120560