GSM8138147: PT4; Homo sapiens; Hi-C
Approximately 100-200 mg of each tissue was chopped into fine pieces before being fixed. Hi-C was performed using Phase Genomics Proximo Hi-C kits v4.0 (Phase genomics, Seattle, WA) according to the manufacturer's instructions21. Briefly, the finely chopped tissues were incubated with crosslinking solution for 15 minutes with gentle rotation. Tissue lysis was performed with motor and pestle with liquid nitrogen and grinded to a fine powder, and resuspended in 700 µl of Lysis buffer 1. The chromatin containing supernatant was pelleted and washed with 500 µl of 1x CRB, resuspended in 100 µl of Lysis Buffer 2. 100 µl of SPRI beads were added, incubated and washed with 200 µl of 1x CRB. SPRI beads containing chromatin were resuspended in 150 µl of fragmentation buffer, 5 µl of restriction enzyme (Sau3AI) and incubated at 37 oC for 1 hour. 2.5 µl of finishing enzyme is added and mixed by vortexing. Ligation was performed by adding 95 µl of Ligation buffer and 5 µl ligation enzyme to the SPRI beads containing chromatin. To revert the crosslinking, 5 µl of RX enzyme was added to the reaction and then incubated overnight at 65 oC with shaking (900 rpm). The pellet was washed, air dried, resuspended in 100 µl of elution buffer and then incubated for 5 minutes at room temperature. The beads were washed and resuspended in 100 µl of bead binding buffer. After the final wash the beads were resuspended in 200 µl of deionized water and measured the concentration of DNA using Qubit dsDNA HS assay kit. About 500 ng of streptavidin beads containing DNA were separated, resuspended in 20 µl of deionized water, and a total of 25 µl of library reagent and an approximate amount of library reagent 1 is added. The reaction was mixed gently and incubated at 55 oC for 10 min. PCR was performed by adding 26 µl of HSR mix (PCR Hotstart Ready mix) and 5 µl of PCR index primer to each reaction. After the amplification, the sample was size selected by adding 25 µl of SPRI beads to the library. The beads were separated and washed twice with 200 µl of 80% ethanol for at least 30 sec each and the pellet was air dried for no longer than 5 min at room temperature. The beads were resuspended and separated and the supernatant containing the final Hi-C library which was quantified using Qubit dsDNA HS assay kit on a Qubit. The library was sequenced on Illumina HiSeq3000.