Hi-C of Homo sapiens: D20 of atrial cardiomyocyte (CM) differentiation with 500 nM dTAGv-1 ligand treated for 24h rep2
TBX5-FKBP12F36V iPSC-CMs were dissociated in 0.25% Trypsin-EDTA for 10 mins, followed by quenching in 15%FBS in DPBS. CMs were lifted off the dish and resuspended to single cells by repeatedly using P1000 pipette and then transferred to a 15 ml tube and centrifuged at 800 rpm for 5 min at room temperature. Pellets were washed once in 5 ml HBSS before resuspending entire cell pellet in exactly 10 ml HBSS. 16% methanol-free formaldehyde was added to a final concentration of 1% and samples incubated on nutator at room temperature for 10 min. Samples were quenched with 2.5 M glycine to a final concentration of 0.125 M and incubated on nutator at room temperature for 5 min, then 15 min at 4C and followed by centrifugation at 1000 x g for 5 min at room temperature. Samples were washed once in DPBS. Pellets were resuspended in 1 ml of 3 mM DSG in PBS, transferred to 1.5 ml tubes and incubated on nutator for 40 min. Samples were quenched with 2.5 M glycine to a final concentration of 0.125 M and incubated on nutator at room temperature for 5 min. Samples were centrifuged at 2000 x g for 5 min at RT. Cell pellets were resuspended in 5 mg/ml BSA in DPBS, to prevent cells sticking to tube after DSG crosslinking, and centrifuged at 2000 x g for 5 min at 4C. Supernatant was carefully removed and cell pellets were snap frozen in liquid nitrogen and stored at -80C. Samples were processed for Hi-C using Arima-HiC+ kit (Arima Genomics). >1 g per sample was used as determined by Arimas Estimating Input Amount Protocol using Qubit Fluorometer and dsDNA HS Assay Kit (ThermoFisher Scientific #Q32854) Samples were processed by following manufacturers user guide for mammalian cell lines with one exception: DNA was digested using 50 U DpnII (New England Biolabs #R0543M) and 50 U DdeI (New England Biolabs #R0175L) restriction enzymes in 1x NEB Buffer 3.1 for 60 min at 37C. All samples passed Arima-QC1 Quality Control. Sequencing libraries were prepared by following Arimas user guide for Library Prep Using KAPA Hyper Prep Kit (Roche #7962347001). Between 1.5-4 g of DNA was fragmented in 130 l microtube (Covaris #520045) using a Covaris S2 sonicator for 55s at 10% duty cycle, intensity 4, 200 cycles per burst in a water bath maintained at 4C. An average fragment size of 400 bp was verified using Agilent 2100 Bioanalyzer. Between 400-1000 ng of size-selected DNA was used for biotin enrichment, end repair, dA-tailing and adapter ligation. The number of PCR cycles was determined following Arima-QC2 Quality Control using KAPA Library Quantification Kit (Roche KK4824) and libraries were amplified to at least 10 nM. All samples passed Arima-QC2 Quality Control. DNA cleanup steps were done using Ampure XP beads (Beckman Coulter #A63881). Final libraries were quantified using Qubit Fluorometer and dsDNA HS Assay Kit and assessed on Agilent BioAnalyzer. Pooled, equimolar libraries were sequenced using NovaSeq technology (Illumina).