GSM6922496: RPE TP53, control, cell 28; Homo sapiens; Hi-C
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. Next, in-nucleus proximity ligation was done with 50U T4 DNA Ligase, 1x T4 DNA ligase buffer, 5% PEG, 1% Triton-X100, 0.1mg/ml BSA at 16°C overnight, with light mixing. Only pools of nuclei with at least 75% of the population showing integral nuclear membrane were considered for further processing. Nuclei were strained multiple times through a 10µm Nylon Net Filter (Merck-Millipore, catalogue no. NY1009000). Sample preparation was done with DispenKit (SEED Bioscience) and single nuclei were dispensed in skirted Eppendorf twin.tec® PCR Plate 96-wells (Eppendorf, catalogue no. 0030128648) containing 50µl of NEBuffer 3.1 (New England Biolabs, catalogue no. B7203S) using the single cell isolator DispenCell (SEED Biosciences) following manufacturer's instructions. A nucleus passing through the tip of the DispenCell leaves an electrical impedance change mark, which is proportional to the volume of the nucleus. For RPE TP53-/- cells, a minimum impedance change of 75Ω was detected for the diploid nucleus and 200Ω for the tetraploid nucleus. A lower impedance change was associated with debris or unsuccessful induction of genome doubling in the case of the WGD condition and thus such nuclei were not considered for further processing. To avoid processing of nuclei aggregates, dispensed single nuclei associated with a threshold higher than 400Ω and 800Ω for diploid and tetraploid nuclei, respectively, were also discarded. These ranges were set from quality metrics of a test scHi-C batch. Specifically, an unreasonable number of unique interactions of each fragment end (e.g. > 2 for diploid loci) would indicate a nuclei aggregate rather than a single nucleus. Following dispensing, the single nuclei were de-crosslinked by incubation at 65°C overnight. Each selected nucleus was mixed with 25µl Dynabeads™ MyOne™ Streptavidin C1 and transferred to a 1.5 ml tube and incubated at room temperature for one hour on a rotating wheel. The bead-bound fragments were digested with 10U of AluI restriction enzyme (New England Biolabs, catalogue no. R0137) in 1x rCutSmart™ Buffer (New England Biolabs, catalogue no. B6004) at 37°C for two hours. 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 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 27-30 cycles. Libraries were cleaned with AMPure XP beads, and then ran on a 2% agarose gel at 100V for 50-60 minutes. Successful libraries presented a 300-700bp smear, which was cut from the gel. DNA purification from the agarose was performed using NucleoSpin Gel and PCR Clean‑up (Macherey-Nagel, catalogue no. 740609) following manufacturer's instructions. An additional AMPure XP bead size selection was generally necessary to remove any primer dimer contamination.