GSM5034249: HCT116-NUP93-mAC,Hi-C,MboI,UT,biological replicate 1; Homo sapiens; Hi-C
5-10 million cells were cross-linked using 1% formaldehyde for 10mins at room temperature, quenched with 0.125M glycine for 10mins at room temperature, and were washed with cold PBS. Cross-linked cell pellets were resuspended in 1ml iced-cold Hi-C lysis buffer (10 mM Tris-HCl, pH 8.0; 10mM NaCl, 0.2% NP-40 and protease inhibitor cocktail), and rotated at 4°C for 30 mins. After pelleting down the nuclei, 100 ul of 0.5% SDS was used to resuspend and permeabilize the nuclei at 62 °C for 10 mins.  Then 260 ul H20 and 50 ul 10% Triton-X100 were added to quench the SDS at 37 °C for 15 mins. Subsequently, enzyme digestion of chromatin was performed at 37 °C overnight by adding an additional 50 ul of 10X NEB buffer 2 and 400U MboI (NEB, R0147M). After overnight incubation, the MboI in solution was inactivated at 62 °C for 20 mins. To fill-in the DNA overhangs and mark the ends with biotin, 35U DNA polymerase I (Klenow, NEB, M0210) together with 10 ul of 1mM biotin-dATP (Jeana Bioscience), 1ul 10mM dCTP/dGTP/dTTP were added and incubated at 37°C for 1 hour with rotation. Blunt end Hi-C DNA ligation was performed using 5000 U NEB T4 DNA ligase with 10X NEB T4 Ligase buffer with 10mM ATP, 90 ul 10% Triton X-100, 2.2 ul 50mg/ml BSA at room temperature for four hours with rotation. After ligation, nuclei were pelleted down and resuspended with 440 ul Hi-C nuclear lysis buffer (50mM Tris-HCl pH7.5; 10mM EDTA, 1% SDS and protease inhibitor cocktail), and further sheared using the parameter of 10/20 secs ON/OFF cycle, 25% Amp, 5mins by a QSonica 800R sonicator. Hi-C will use 5-10% of the sonicated chromatin in the next section. For HiChIP, the sonicated chromatin was incubated with appropriate antibody (here by 4ug H3K27ac polyclonal antibody) overnight, and the immunocomplex was captured by dynabeads protein G in the next day. The immunocomplex was washed 2 times by low salt wash buffer (20 mM Tris-HCl pH 7.5, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), 2 times by high salt wash buffer (20 mM Tris-HCl pH 7.5, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl), 1 time with Tris-EDTA buffer + 0.1% triton-100, and one more time with TE only. The immunocomplex was subjected to overnight decrosslinking at 65°C, protein K treatment, and DNA extraction For both Hi-C and HiChIP library making , after DNA extraction, they were used for biotin DNA enrichment by 20 ul Dynabeads® MyOne™ Streptavidin C1 beads (Thermo Fisher 65002). The biotin bound DNA on beads was used to perform on-beads library making with KAPA Hyper DNA library kit, or using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (NEB, E7645L).
GEO Accession
GSM5034249