GSM9746001: hiPSC-derived Cardiomyocyte, day10, biol rep 2; Homo sapiens; ATAC-seq
Engineered heart tissues were pooled from 2x 48 well plates and incubated at 37° C for 1.5-2 hours in a dissociation solution containing 1mg/ml Collagenase II,10 mM HEPES pH 7.4, 2 mM Thiazovivin, and 30 mM N-Benzyl-p-Toluenesulfonamide to obtain a single cell suspension. Cells were filtered through 70 um cell strainer. Cells were sorted for eGFP expression using fluorescence-assisted cell sorting to generate eGFP+ cardiomyocyte fractions and eGFP- cardiac fibroblast fractions for library preparation. Input cardiomyocytes were dissociated for 1-2 hours in a dissociation solution containing 200 units / ml Collagenase IV,10 mM HEPES pH 7.4, 2 mM Thiazovivin, and 30 mM N-Benzyl-p-Toluenesulfonamide and passed through a 70 um cell strainer. Cardiomyocytes were isolated using column-based purification before library preparation. Sorted cells were washed again with 10mL EHT media and resuspended in 1mL with 2%BSA. The cells were then counted and separated into aliquots of 50,000. Assay for transposase-accessible chromatin sequencing (ATAC-Seq) was performed as described by Grandi, F. C. et al. 2022. Briefly, 50k cells were collected at 500g for 5min at 4°C and gently resuspended in 50 ml ice-cold lysis buffer: 10 mM Tris–HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin. Lysis proceeded for 3min on ice and was neutralized with 1ml of ice-cold lysis buffer excluding detergents digitonin and NP40. Following neutralization, permeabilized nuclei were pelleted at 4°C and resuspended in 50µl transposition master mix: 25µl (2X) Illumina TD buffer, 16.5 ml PBS, 5 ml H2O, 0.5 ml 1% digitonin, 0.5 ml 10% Tween-20, and 2.5 ml Illumina TDE1 enzyme, and incubated for 30min at 37°C on a thermomixer. Tagmentation was terminated and DNA collected using Zymo Clean and Concentrator-5 following the manufacturers recommendations. Libraries were barcoded with 5 cycles of preamplification, and the PCR reaction was paused for quantification using NEBNext Library Quant Kit for Illumina. Diluted final libraries were mixed and sequenced on an Illumina Nova-Seq 6000 using paired end 50bp reads.