title: GSM4140487: Untreated_T0_HiC_run1; Homo sapiens; Hi-C
library_construction_protocol: Adherent cells were directly cross-linked on the plates with 1% formaldehyde for 10min at room temperature. After addition of glycine (125mM final) to stop the reaction, cells were washed with PBS and recovered by scrapping. Cross-linked cells were incubated 30min on ice in 3C lysis Buffer (10mM Tris-HCl pH 8, 10mM NaCl, 0.2% NP40, 1X anti-protease cocktail), centrifuged 5min at 3,000 rpm and resuspended in 190uL of NEBuffer2 1X  (New England Biolabs – NEB). 10 µL of 10% SDS were added and cells were incubated for 10min at 65ºC. After addition of Triton X-100 and 15min incubation at 37ºC, nuclei were centrifuged 5min at 3,000 rpm and resuspended in 300uL of NEBuffer2 1X. Digestion was performed overnight using 400 U MboI restriction enzyme (NEB). To fill-in the generated ends with biotinylated-dATP, nuclei were pelleted and resuspended in fresh repair buffer 1x (1.5µL of 10mM dCTP; 1.5µL of 10mM dGTP; 1.5µL of 10mM dTTP; 37.5µL of 0.4mM Biotin-dATP; 50U of DNA Polymerase I Large (Klenow) fragment in 300uL NEBuffer2 1X). After 45min incubation at 37ºC, nuclei were centrifuged 5min at 3,000 rpm and ligation was performed 4h at 16ºC using 10,000 cohesive end units of T4 DNA ligase (NEB) in 1.2mL of ligation buffer (120µL of 10X T4 DNA Ligase Buffer; 100µL of 10% Triton X-100; 12µL of 10mg/mL BSA; 963µL of H2O). After reversion of the cross-link, DNA was purified by phenol extraction and EtOH precipitation. Purified DNA was sonicated to obtain fragments of an average size of 300-400 bp using a Bioruptor Pico (Diagenode; 8 cycles; 20s on and 60s off). 3µg of sonicated DNA was used for library preparation. Biotinylated DNA was pulled down using 20uL of Dynabeads Myone T1 streptavidine beads in Binding Buffer (5mM Tris-HCl pH7.5; 0.5mM EDTA; 1M NaCl). End-repair and A-tailing were performed on beads using NEBnext library preparation end-repair and A-tailing modules (NEB). Illumina adaptors were ligated and libraries were amplified by 8 cycles of PCR. Resulting Hi-C libraries were first controlled for quality by low sequencing depth on a NextSeq500 prior to higher sequencing depth on HiSeq2000.
attributes.tag: GEO Accession
attributes.value: GSM4140487