Illumina HiSeq 4000 paired end sequencing; Promoter capture and whole genome in situ Hi-C of human iPSC and iPSC-derived cardiomyocytes from an Yoruban individual (NA19101)
Promoter capture and whole genome in situ Hi-C of human iPSC and iPSC-derived cardiomyocytes from an Yoruban individual (NA19101)
We used the iPSC line NA19101 (https://www.coriell.org/0/Sections/Search/Sample_Detail.aspx?Ref=GM19101&product=CC) to generate cardiomyocytes. Cardiomyocyte differentiations were carried out similar to the protocol of Burridge et al, 2013 with slight modifications. Briefly, we initiated mesoderm differentiation of the iPSCs with the GSK-3 inhibitor CHIR-99021 trihydrochloride for 24 hours (Tocris #4953). Two days later, cardiomyocyte differentiation was initiated with 2μM Wnt-C59 (Tocris #5148) for 48 hours. Cells exhibited spontaneous beating by day 8-10 and were subsequently selected with lactate on days 15-20 at which time they were harvested and analyzed with flow cytometry for cardiac troponin T. We followed the protocol of Rao et al. 2014 (doi: 10.1016/j.cell.2014.11.021.) to make in situ Hi-C libraries: All cell lines were cultured following the manufacturer's recommendations. Two to five million cells were crosslinked with 1% formaldehyde for 10 min at room temperature. Nuclei were permeabilized. DNA was digested with 100 units of MboI, and the ends of restriction fragments were labeled using biotinylated nucleotides and ligated in a small volume. After reversal of crosslinks, ligated DNA was purified and sheared to a length of ∼400 bp, at which point ligation junctions were pulled down with streptavidin beads and prepped for Illumina sequencing. For each Hi-C library, we used 5 million cells and the MboI restriction enzyme. Pre-capture Hi-C libraries were amplified with 6 PCR cycles and post-capture Hi-C libraries were amplified with 8 PCR cycles. The capture probes were designed to target the ends of MboI fragments at least 1kb apart surrounding all RefSeq genes, with four baits per gene.
Experimental Factor: cell type
cardiac myocyte