GSM6788425: iPSC_HiC; Homo sapiens; Hi-C
We fixed the cells for all downstream Hi-C assays as previously described (Beagan et al., 2017; Beagan et al., 2016; Beagan et al., 2020; Phillips-Cremins et al., 2013). Briefly, we fixed cells by adding 1 % (v/v) formaldehyde in DMEM/F-12 (Thermo Fisher, 11320033) for 10 min at room temperature. The complete fixation media was 50 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 11 % formaldehyde. We quenched the fixation process by adding 125 mM glycine for 5 min at room temperature, following by 15 min at 4 °C. We washed the cross-linked cells in pre-chilled PBS before flash freezing and stored the pellets at −80 °C. We used ~2 million cross-linked cells per replicate per condition and processed two biological replicates per condition. We used Arima-HiC kit (Arima Genomics, Inc.; Cat # A510008) for Hi-C sample preparation according to the manufacturer's recommended protocol. Arima-HiC protocol utilizes multiple restriction enzymes to digest chromatin. Following restriction of the chromatin, we filled in the 5'-overhangs to label the digested ends with a biotinylated nucleotide.  Next, we ligated spatially proximal digested ends of DNA and purified the proximally-ligated DNA. We prepared samples for library preparation by shearing purified proximally-ligated DNA. We sheared to an average size of ~400 bp using a Covaris S220 sonicator at 140 W peak incident power, 10% duty factor, and 200 cycles per burst for 55 seconds. We performed size-selection to obtain 200-600 bp DNA fragments using DNA Purification Beads (AMPure XP Beads, Beckman Coulter, A63881). We enriched biotin-tagged ligation junctions after size-selection using Enrichment Beads (provided in Arima-HiC kit). We stored streptavidin beads containing enriched DNA fragments up to 3 days before proceeding with the library preparation step. We prepared all sequencing libraries using the NEBNext Ultra II Library Prep Kit (NEB, E7645S) following the manufacturer's protocol. We first end repaired, and dA-tailed DNA from HiC experiments and ligated with Illumina adaptors using NEBNext Ultra II DNA Library Prep Kit for Illumina according to the manufacturer's protocol with minor modifications. We washed adaptor-ligated Hi-C libraries on streptavidin beads twice in 150 μl of wash buffer at 55 °C and once in 100 μl of elution buffer (Arima Genomics, A510008), and then eluted ligation products from streptavidin beads by boiling at 98 °C for 10 min in 15 μl elution buffer. For ChIP-seq, we size-selected adaptor-ligated libraries using AgenCourt Ampure XP beads (Beckman Coulter, A63881) and then amplified using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S) according to the manufacturer's protocol. We sequenced Hi-C libraries with reading length 37 bp pair-ended.