GSM5570626: Diff_rep1; Homo sapiens; Hi-C
For the promoter Capture Hi-C,, cells were rinsed with serum-free media prior to fixation. We fixed the cells directly in culture plates with 2% formaldehyde (EMD Millipore 344198) in serum-free media (PromoCell). We incubated cells in fixation medium with rocking at room temperature for 10 minutes, and then quenched with 1 M ice-cold glycine for a final concentration of 125 mM. After 5 minutes of rocking incubation at room temperature, we rinsed fixed cells twice with ice-cold PBS. Then we incubated the cells with rocking on ice with ice-cold permeabilization buffer (10 mM Tris–HCl pH 8, 10 mM NaCl, 0.2 % Igepal CA-630, Complete EDTA-free protease inhibitor cocktail [Roche])36 for 30 minutes. We scraped cells from the culture plate and centrifuged at 2500 x g for 5 minutes at 4ᵒC to pellet nuclei. The supernatant was discarded and nuclei were flash frozen in liquid nitrogen and put at -80ºC. The Hi-C library was prepared as described in (Rao et al., Cell, 2014) with modifications. We processed nuclei in 5M cell aliquots, closely following Rao et al. protocol I.a.1 except we digested chromatin with 400U of HindIII (New England Biolabs R3104) at 37ºC overnight with shaking (950 rpm). After digestion, we pelleted nuclei with centrifugation at 2500 x g for 5 minutes at 4ºC. We performed end-filling and marked with biotin using biotin-14-dCTP (Invitrogen 19518-018), performed in-nucleus ligation, degraded protein using Proteinase K, and performed ethanol precipitation and purification. We sheared 5 μg of DNA to 250-550 bp using the Covaris M220 instrument. We performed double size-selection to retain fragments from 250-550 bp using Agencourt AMPure XP beads (Beckman Coulter A63881). We immobilized the fragments containing biotin using DYNAL™ MyOne™ Dynabeads™ Streptavidin T1 (Invitrogen 65601) beads. After end-repair and attachment of dATP, we ligated Illumina paired-end adaptors to the bead-bound library following the SureSelectXT user manual for Illumina Paired-End Multiplexed Sequencing (Agilent Technologies). After washing, we resuspended the Hi-C library in 20 μl of 1x Tris buffer and subsequently removed the Streptavidin beads from the DNA by heating at 98ºC for 10 minutes. We then amplified the adaptor-ligated library using 8 PCR cycles and purified using Agencourt AMPure XP beads, following the SureSelectXT user manual. The RNA baits for promoter capture were designed in (Mifsud et al., Nature Genetics, 2015) for capturing HindIII fragments containing gene promoters (Dr. Cameron Osborne kindly shared the exact design). A total of 550 ng of the Hi-C library was hybridized to the biotinylated RNA baits, captured with DYNAL™ MyOne™ Dynabeads™ Streptavidin T1 beads, and amplified in a post-capture PCR to add indexes, using 6 PCR cycles. The library was sequenced on the Illumina HiSeq 4000 platform. Promoter Capture Hi-C