GSM1603856: GM12878 Rep1; Homo sapiens; OTHER
In preparation for Hi-C assay, cells were pelleted and resuspsended in media supplemented with formaldehyde at a final concentration of 1% for chromatin crosslinking. Crosslinking was halted in ~200mM Glyceine, pelleted, washed with cold PBS, pelleted, snap frozen, and stored at -80 until experimentation. Hi-C libraries were prepared using the published method (Lieberman-Aiden, Science, 2009). Briefly, cross-linked cell pellets were lysed and chromatin within intact nuclei were digested using HindIII. Digested ends were filled in using biotinylated dCTP and an E. coli DNA Pol I large fragment (Klenow polymerase). Blunt-ended DNA molecules were then proximally ligated using T4 DNA ligase. After chromatin cross-linking was reversed, ligated DNA was purified. Next, purified ligation products were treated with T4 DNA polymeras, dATPs and dGTPs to remove biotinylated dCTPs from unligated DNA molecules and column-purified. Then, ligation products were fragmented to ~400bp using a Covaris S2 instrument. Ends of fragmented molecules were then repaired using a combination of T4 DNA polymerase, Klenow polymerase and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (exo minus) and dATP to yield a protruding 3- 'A' base for eventual ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. Yet after dA-tailing, DNA molecules were size selected on agarose gel from 200-600bp. After gel purification, biotinylated DNA molecules were pulled down using C1 streptavidin beads (Life Tech) and Illumina sequencing adapters were ligated using T4 DNA ligase. The bead-bound library was then PCR amplified using 14-15 cycles and purified using AMPure XP beads prior to target enrichment. For target enrichment, 500ng of Hi-C library was mixed with 500ng of RNA probes and incubated overnight at 65 degrees. The following day, captured DNA:RNA duplexes were bound to T1 streptavidin-coated beads (Life Tech), washed rigorously, and PCR amplified using 10-11 cycles. Final PCR amplicons were purified using AMPure XP beads and captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the HiSeq2500 following the manufacturer's protocols for 100 cycle, paired-end sequencing.
GEO Accession
GSM1603856