title: GSM6922435: CP-A TP53-/-, clone 3, spontaneous high ploidy, rep1; Homo sapiens; Hi-C
library_construction_protocol: 1-2 million cells were collected and fixed with 2% formaldehyde (Thermo Fisher Scientific, catalogue no. 11483217). The reaction was quenched with 200mM glycine (VWR, catalogue no. 101194M) final concentration, cells were washed with PBS (Thermo Fisher Scientific, catalogue no. 10010023) and lysed in a solution containing 10mM Tris-HCl pH 8.0 (Thermo Fisher Scientific, catalogue no. 15568025), 10mM NaCl (Sigma-Aldrich, catalogue no. S6546), 0.2% Igepal CA-630 (Sigma-Aldich, catalogue no. I8896), and 1x Proteinase inhibitor cocktail (Roche, catalogue no. 11697498001) at 4°C for 30 minutes. Resulting nuclei were re-suspended in 1X NEB3.1 buffer (New England Biolabs, catalogue no. B7203S). Nuclei suspension was incubated with 0.11% SDS (Carl Roth, catalogue no. CN30) final concentration for 10 minutes at 65°C. Reaction was quenched with 1% Triton-X100 (AppliChem, catalogue no. A1388) and nuclei were digested with 100U MboI restriction enzyme (New England Biolabs, catalogue no. R0147) at 37°C overnight. Restriction enzyme was inactivated according to manufacturer's specifications and digested nuclei were washed and re-suspended in 1x NEB3.1. Digested ends were then marked with biotin via incubation in 0.03mM biotin-14-dATP (Thermo Fisher Scientific, catalogue no. 19524016), 0.03mM dCTP, 0.03mM dGTP, 0.03mM dTTP (Promega, catalogue no. U1420) and 50U Klenow DNA polymerase I (New England Biolabs, catalogue no. M0210M) for 4 hours at room temperature. Resulting blunt-ends were proximally ligated with 50U T4 DNA Ligase (Thermo Fisher Scientific, catalogue no. EL0011), 1x T4 DNA ligase buffer (Thermo Fisher Scientific, catalogue no. B69), 5% PEG, 1% Triton-X100, 0.1mg/ml BSA (New England Biolabs, catalogue no. B9000S) for 4 hours at room temperature. Crosslink reversal was performed on the proximity-ligated chromatin through the incubation with 300mM NaCl and 1% SDS overnight, at 68°C. The sample was then treated with 50g/ml RNase A (Thermo Fisher Scientific, catalogue no. EN0531) for 30 minutes at 37°C, followed by 400ug/ml Proteinase K (Promega, catalogue no. V3021) at 65°C for one hour. DNA was purified via precipitation with 1.6 volumes of pure ethanol and 0.1 volumes of sodium acetate, pH 5.2 (Thermo Fisher Scientific, catalogue no. R1181) at -80°C. DNA was eluted and then fragmented by sonication at 80V peak incidence power, 10% duty factor, 200 cycles per burst, for 60-80 seconds with an E220 Focused-ultrasonicator (Covaris). Sheared DNA was size selected for library preparation using AMPure XP beads (Beckman Coulter, catalogue no. A63881). Biotin-marked fragments were isolated with Dynabeads™ MyOne™ Streptavidin C1 (Thermo Fisher Scientific, catalogue no. 65001) and all subsequent steps were performed on the bead-bound DNA fraction. Hi-C library preparation continued with end polishing reaction, which involved incubation of DNA with 1x T4 ligase buffer (New England Biolabs, catalogue no. B0202S), 2.5mM each dNTP, 50U T4 Polynucleotide Kinase (New England Biolabs, catalogue no. M0201), 12U T4 DNA Polymerase (New England Biolabs, catalogue no. M0203), and 5U Klenow DNA polymerase I, at room temperature for 30 minutes. PolyA tail was added by incubating the DNA sample in 1X NEBuffer™ 2 (New Enlgand Biolabs, catalogue no. B7002S) with 0.5mM dATP and 25U Klenow Fragment (3'→5' exonuclease) (New England Biolabs, catalogue no. M0212) at 37°C for 30 minutes. DNA fragment ends were then ligated to Illumina TruSeq unique dual indexes (Integrated DNA Technologies) in 1X T4 Ligation buffer with 5% PEG and 15U T4 DNA Ligase for 2 hours at room temperature or overnight at 16°C. Lastly, libraries were PCR amplified using Illumina forward (AATGATACGGCGACCACCGAGATCTACAC) and reverse (CAAGCAGAAGACGGCATACGAGAT) primers and KAPA HiFi HotStart ReadyMix (Roche, catalogue no. KK2602) for 6-10 cycles. Resulting fragments were size-selected with AMPure XP beads.