GSM3067222: STL003.SX3.SRC1007; Homo sapiens; OTHER
Promoter Capture Hi-C library was constructed by performing a target enrichment protocol (enriching target promoter-centered proximity ligation fragments from Hi-C library using capture RNA probes). Briefly, we incubated 500ng Hi-C library 24h at 65 °C within a humidified hybridization chamber with 2.5ug human Cot-1 DNA (Life Technologies), 2.5ug salmon sperm DNA (Life Technologies), and p5/p7 blocking oligos with hybridization buffer mix (10X SSPE, 10mM EDTA, 10X Denhardts solution, and 0.26% SDS) and 500ng RNA probes. RNA:DNA hybrids were enriched using 50ul T1 streptavidin beads (Invitrogen) with 30min incubation at RT. Next, bead-bound hybrids were washed by a 15min incubation in wash buffer1 (1X SSC and 0.1% SDS) with frequent vortexing, and then washed three times with 500ul of pre-warmed (65 °C) wash buffer2 (0.1X SSC and 0.1% SDS), and resuspended in nuclease-free water. The resulting capture Hi-C libraries were amplified while bound to the T1 beads, and purified using AMPure XP beads, followed by sequencing. Promoter Capture Hi-C library sequencing procedures were carried out as previously described according to Illumina HiSeq2500 or HiSeq4000 protocols with minor modifications (Illumina, San Diego, CA). RNA-seq experiments were conducted using Ovation® RNA-Seq System V2 (NuGEN) as previously described (Dahl et al., 2016 Nature).