GSM7995218: SH-SY5Y, WT, 5 Day ATRA Treatment, Rep 2; Homo sapiens; Hi-C
For each Hi-C library, approximately 5 million cells were used as input. Cells were crosslinked for 10 minutes at room temperature in a final concentration of 1% formaldehyde. Glycine was added to quench crosslinking. Cells were then crosslinked with 3mM DSG for 45 minutes at room temperature, and DSG crosslinking was quenched with Tris. Cell pellets were frozen at -80C until Hi-C library generation. Flash-frozen cross-linked cells were thawed on ice for Hi-C, and were then lysed and digested with DpnII following the Hi-C 2.0 protocol (Belaghzal et al., 2017). Crosslinked chromatin was digested with DpnII (NEB, R0543M) at 37 degrees C overnight, following the Hi-C 2.0 protocol (Belaghzal et al., 2017). The overhanging DNA ends were filled in using biotin-14-dATP (LifeTech, 19524016) at 23 degrees C for four hours, and ligated with T4 DNA ligase (Life Technologies, 15224090) at 16 degrees C for four hours. Chromatin was then treated with proteinase K (ThermoFisher, 25530031) at 65 degrees C overnight to remove all cross-linked proteins. Ligation products were purified by phenol:chloroform extraction with ethanol precipitation, fragmented by sonication, and size-selected using SPRI beads to retain fragments of 100-350bp. We performed end repair and then selectively purified biotin-tagged DNA using streptavidin coated beads (DYNAL™ MyOne™ Dynabeads™ Streptavidin C1, Invitrogen 65001). A-tailing and Illumina TruSeq adapter ligation (Illumina, 20015964) were performed on the bead-bound ligation products, and samples were amplified using the TruSeq Nano DNA Sample Prep kit (Illumina, 20015964). PCR primers were removed using SPRI beads (1.1x ratio) before sequencing the final Hi-C libraries using PE50 bases on an Illumina HiSeq 4000.