GSM6031777: C28_HiC_1.2; Homo sapiens; Hi-C
C-28/I2 cells were cultured in DMEM/F12 media with 10% fetal bovine serum, 0.1% penicillin streptomycin solution, 0.1% amphotericin B, and 0.04% gentamicin. Cells were treated with DMEM/F12 media with 1% ITS-Plus for 48 hours prior to experiments to promote the chondrocyte phenotype. Cells were then washed with 1X PBS and treated with trypsin-EDTA (0.25%) for 3 minutes. Trypsin was quenched and cells were pelleted at 4°C for 5 minutes at 300g. Cells were resuspended in 1mL DMEM/F12 per million cells and crosslinked in 1% formaldehyde for 10 min with rotation before quenching in a final concentration of 0.2M glycine for 5 min with rotation. Cells were pelleted at 300g for 5 min at 4°C. Pellets were washed with cold PBS and aliquoted into ~3 million cell aliquots. Pellets were flash frozen in liquid nitrogen and stored at -80°C. In situ Hi-C was performed as previously described42. Pellets were lysed in ice-cold Hi-C lysis buffer (10mM Tris-HCl pH 8.0, 10mM NaCl, 0.2% IGEPAL CA630) with 50μL of protease inhibitors for 15 min on ice. Cells were pelleted and washed using the same buffer. Pellets were resuspended in 50μL 0.5% SDS and incubated at 62°C for 7 min. Reactions were quenched with 145μL water and 25μL 10% Triton X-100 at 37°C for 15 min. Chromatin was digested overnight with 25μL 10X NEBuffer2 and 100U MboI at 37°C with rotation. Reactions were incubated at 62°C for 20 min then cooled to RT. Fragment overhangs were repaired by adding 37.5μL 0.4mM biotin-14-dATP; 1.5μL each 10mM dCTP, dGTP, dTTP; 8μL 5U/μL DNA Polymerase I, Large (Klenow) Fragment and incubating at 37°C for 1.5 h with rotation. Ligation was performed by adding 673μL water, 120μL 10X NEB T4 DNA ligase buffer, 100μL 10% Triton X-100, 6μL 20mg/mL BSA, and 1μL 2000U/μL T4 DNA ligase and incubating at RT for 4 h with slow rotation. Samples were pelleted at 2500g, resuspended in 432μL water, 18μL 20mg/mL proteinase K, 50μL 10% SDS, and 46μL 5M NaCl, incubated at 55°C for 30 min, and then transferred to 68°C overnight. Samples were cooled to RT and 1.6x volumes of pure ethanol and 0.1x volumes of 3M sodium acetate pH 5.2 were added to each sample, prior to incubation at -80°C for over 4-6 h. Samples were spun at max speed at 2°C for 15 min and washed twice with 70% ethanol. Pellets were dissolved in 130μL 10mM Tris-HCl pH 8.0 and incubated at 37°C for 1-2 h. Samples were stored at 4°C overnight. DNA was sheared using the Covaris LE220 (Covaris, Woburn, MA) to a fragment size of 300-500bp in a Covaris microTUBE. DNA was transferred to a fresh tube and the Covaris microTUBE was rinsed with 70μL of water and added to the sample. A 1:5 dilution of DNA was run on a 2% agarose gel to verify successful shearing. Sheared DNA was size selected using AMPure XP beads. 0.55x volumes of 2X concentrated AMPure XP beads were added to each reaction and incubated at RT for 5 min. Beads were reclaimed on a magnet and the supernatant was transferred to a fresh tube. 30μL 2X concentrated AMPure XP beads were added and incubated for 5 min at RT. Beads were reclaimed on a magnet and washed with fresh 70% ethanol. Beads were dried for 5 min at RT prior to DNA elution in 300μL 10mM Tris-HCl pH 8. Undiluted DNA was run on a 2% agarose gel to verify successful size selection between 300-500 bp. 150μL 10mg/mL Dynabeads MyOne Streptavidin T1 beads were washed with 400μL 1X Tween washing buffer (TWB; 250μL Tris-HCl pH 7.5, 50μL 0.5M EDTA, 10mL 5M NaCl, 25μL Tween 20, 39.675μL water). Beads were then resuspended in 300μL 2X Binding Buffer (500μL Tris-HCl pH 7.5, 100μL 0.5M EDTA, 20mL 5M NaCl, 29.4mL water), added to the DNA sample, and incubated at RT for 15 min with rotation. DNA-bound beads were then washed twice with 600μL 1X TWB at 55°C for 2 min with shaking. Beads were resuspended in 100μL 1X NEBuffer T4 DNA ligase buffer, transferred to a new tube, and reclaimed. Sheared ends were repaired by resuspending the beads in 88μL 1X NEB T4 DNA Ligase Buffer with 1mM ATP, 2μL 25mM dNTP mix, 5μL 10U/uL NEB T4 PNK, 4uL 3U/uL NEB T4 DNA polymerase I, and 1uL 5U/uL NEB DNA polymerase 1, large (Klenow) fragment and incubating at RT for 30 min. Beads were washed two more times with 1X TWB for 2 min at 55°C with shaking. Beads were washed once with 100uL 1X NEBuffer 2, transferred to a new tube, and resuspended in 90uL 1X NEBuffer 2, 5uL 10mM dATP, and 5uL NEB Klenow exo minus, and incubated at 37°C for 30 min. Beads were washed two more times with 1X TWB for 2 min at 55°C with shaking. Beads were washed in 100uL 1X Quick Ligation Reaction Buffer, transferred to a new tube, reclaimed, and resuspended in 50uL 1X NEB Quick Ligation Reaction Buffer. 2uL NEB DNA Quick Ligase and 3uL of an appropriate Illumina indexed adapter (TruSeq nano) were added to each sample before incubating at RT for 15 minutes. Beads were reclaimed and washed twice with 1X TWB for 2 min at 55°C. Beads were washed in 100uL 10mM Tris-HCl pH 8, transferred to a new tube, reclaimed, and resuspended in 50uL 10mM Tris-HCl pH 8. Hi-C libraries were amplified directly off T1 beads with 8 cycles in 5uL PCR primer cocktail, 20uL Enhanced PCR mix, and 25uL of DNA on beads. The PCR settings were as follows: 3 min at 95°C followed by 4-12 cycles of 20s 98°C, 15s at 60°C, and 30s at 72°C. Samples were held at 72°C for 5 min before holding at 4°C. Amplified samples were transferred to a new tube and brought to 250uL in 10mM Tris-HCl pH 8. Beads were reclaimed and the supernatant containing the amplified library was transferred to a new tube. Beads were resuspended in 25uL 10mM Tris-HCl pH 8 and stored at -20°C. 0.7x volumes of warmed AMPure XP beads were added to the supernatant sample and incubated at RT for 5 min. Beads were reclaimed and washed with 70% ethanol without mixing. Ethanol was aspirated. Beads were resuspended in 100uL 10mM Tris-HCl pH 8, 70uL of fresh AMPure XP beads were added, and the solution was incubated for 5 min at RT. Beads were reclaimed and washed twice with 70% ethanol without mixing. Beads were left to dry and DNA was eluted in 25uL 10mM Tris-HCl pH 8. The resulting libraries were then quantified by Qubit and Tapestation. A low depth sequence was performed first using the Miniseq sequencer system (Illumina) and analyzed using the Juicer pipeline to assess quality. The resulting libraries underwent paired-end 2x150bp sequencing on an Illumina NovaSeq sequencer. Each replicate was sequenced to an approximate depth of 750 million reads. The full sequencing depth was 2.8 billion reads.