GSM8620849: Thap6h_HiC; Mus musculus; Hi-C
Hi-C 3.0 was performed performed as previously described with some modifications. Briefly, 5 million cells were washed once with ice-cold PBS and then crosslinked with 1% formaldehyde for 10 min at room tempreture. Crosslinking was quenched with 0.2 M glycine for 5 min. Cells were further crosslinked with 3 mM DSG for 40 min and quenched again with 0.4 M glycine for 5 min. Crosslinked cell pellets were washed with cold PBS and then resuspended in 0.5 ml of ice-cold Hi-C lysis buffer and rotated at 4 °C for 30 min. After washing with 0.5 ml of ice-cold Hi-C lysis buffer, nuclei were resuspend in 0.5% SDS to open up the chromatin at 65°C for 10 min and quenched with Triton X-100 at the final concentration of 1%. Subsequently, enzyme digestion of chromatin was performed at 37 °C overnight by adding 50 μl of 10X NEB buffer 2, 400 U of DpnⅡ(NEB #R0543M) and 400 U of DdeI (NEB#R0175L). After overnight incubation, the restriction enzymes were inactivated at 62 °C for 20 min. To fill in the DNA overhangs and add biotin tags, 35 U of DNA polymerase I (Klenow, NEB# M0210L), 10 μl of 1 mM biotin-14-dATP (Thermo fisher #19524016) and 1 μl of 10 mM dCTP/dGTP/dTTP were added and incubated at 37 °C for 4 h with interval shaking (shaked at 950 rpm for 10 s with 5-min intervals). Chromatin were then ligated with T4 DNA ligase ((NEB #M0202M) ) at 16°C for 4 hours, and de-crosslinked by proteinase K treatment, followed by phenol/chloroform extraction coupled with isopropanol precipitation using glycogen as a carrier. Then DNAs were sonicated using a Covaris S220 to approximately 300-500 bp, and biotin-labelled DNAs were purified by Dynabeads MyOne streptavidin C1 beads (Thermo Fisher #65002). The DNA libearies were further prepared for sequencing using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme # ND607) and barcoded adaptors were ligated using TruePrep Index Kit V2 for Illumina (Vazyme #TD202). Size selection was carried out using the VAHTS DNA Clean Beads (Vazyme #N411). Library quantity was assessed Qubit double-stranded DNA (dsDNA) High-Sensitivity Assay. Hi-C 3.0 was performed performed as previously described with some modifications. Briefly, 5 million cells were washed once with ice-cold PBS and then crosslinked with 1% formaldehyde for 10 min at room tempreture. Crosslinking was quenched with 0.2 M glycine for 5 min. Cells were further crosslinked with 3 mM DSG for 40 min and quenched again with 0.4 M glycine for 5 min. Crosslinked cell pellets were washed with cold PBS and then resuspended in 0.5 ml of ice-cold Hi-C lysis buffer and rotated at 4 °C for 30 min. After washing with 0.5 ml of ice-cold Hi-C lysis buffer, nuclei were resuspend in 0.5% SDS to open up the chromatin at 65°C for 10 min and quenched with Triton X-100 at the final concentration of 1%. Subsequently, enzyme digestion of chromatin was performed at 37 °C overnight by adding 50 μl of 10X NEB buffer 2, 400 U of DpnⅡ(NEB #R0543M) and 400 U of DdeI (NEB#R0175L). After overnight incubation, the restriction enzymes were inactivated at 62 °C for 20 min. To fill in the DNA overhangs and add biotin tags, 35 U of DNA polymerase I (Klenow, NEB# M0210L), 10 μl of 1 mM biotin-14-dATP (Thermo fisher #19524016) and 1 μl of 10 mM dCTP/dGTP/dTTP were added and incubated at 37 °C for 4 h with interval shaking (shaked at 950 rpm for 10 s with 5-min intervals). Chromatin were then ligated with T4 DNA ligase ((NEB #M0202M) ) at 16°C for 4 hours, and de-crosslinked by proteinase K treatment, followed by phenol/chloroform extraction coupled with isopropanol precipitation using glycogen as a carrier. Then DNAs were sonicated using a Covaris S220 to approximately 300-500 bp, and biotin-labelled DNAs were purified by Dynabeads MyOne streptavidin C1 beads (Thermo Fisher #65002). The DNA libearies were further prepared for sequencing using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme # ND607) and barcoded adaptors were ligated using TruePrep Index Kit V2 for Illumina (Vazyme #TD202). Size selection was carried out using the VAHTS DNA Clean Beads (Vazyme #N411). Library quantity was assessed Qubit double-stranded DNA (dsDNA) High-Sensitivity Assay. HiC3.0