GSM7635698: CT27, EVT, rep2; Homo sapiens; Hi-C
Cells were rinsed with 5 mL of PBS and then 5 mL PBS containing 2% formaldehyde were added and incubated for 10 min. Then, 550 μL of glycine 1.25 M (final conc 0.125M) were added and incubated for 5 min at room temperature before an additional 15 min incubation on ice. Liquid was removed and cells were rinsed two times with 5 mL ice-cold PBS. Cells were scraped and transferred into 2 mL low bind tubes to have the equivalent of 1*10^6 cells (1 Petri Dish). Nuclei were pelleted at 2,500 rpm for 10 min at 4˚C (repeat if necessary) and all supernatant was removed, and flash frozen on dry ice and stored at -80˚C until ready for use. Crosslinked samples were thawed on ice and prepared for Hi-C using the Arima-HiC kit (Arima Genomics, San Diego, CA). Cross-linking was performed according to the Arima-HiC protocol with the following modifications. At step 5, the incubation at 37°C was increased to a duration of 60 min and the samples were maintained overnight at 4°C after step 11. Samples were then cleaned with AMPure XP Beads as described in the protocol and all quantification and quality control steps that required the use of a Qubit were performed using a Qubit dsDNA HS Assay Kit. Proximally ligated DNA (1000 ng/sample) was used as an input into the library preparation protocol. Samples were sheared to 400 bp using a Covaris LE220-plus system using the manufacturer's recommended settings. Sizing was confirmed using a High Sensitivity DNA Bioanalyzer kit. Size-selection as described in the Arima-HiC protocol was followed and the entire size-selected sample was used as input into biotin enrichment and library preparation with a KAPA HyperPrep kit and Roche SeqCap Adapters (7141530001 or 7141548001, Roche). Instead of performing the Arima-HiC quality control step 2, the number of cycles needed for library amplification was determined as follows: perform 10 PCR cycles for 125-200 ng of input, perform 9 PCR cycles for 200-400 ng of input, perform 8 PCR cycles for 400-600 ng of input, and perform 7 cycles of PCR for inputs greater than 600 ng. The completed libraries were then quantified using a Qubit dsDNA BR Assay Kit and the library size was determined with a High Sensitivity DNA Bioanalyzer Kit. Standard Illumina Free-Adapter Blocking was performed on the libraries. Cleaned, adapter-blocked libraries were loaded on a NovaSeq6000 with a run configuration of 151x8x8x151 and an average depth of 900M PE reads per library.