GSM6043947: Replicate 3; BRD4884 treated; Day 5 differentiated hiPSCs; Homo sapiens; ATAC-seq
Omni-ATACseq sequencing libraries were generated from Day 5 cells undergoing hiPSC cardiac-directed differentiation using an adapted published workflow (Corces et al., 2017). Briefly day 5 cells were harvested and 15,000 cells were pelleted at 500 x g for 5 min at 4oC and washed once in 50 µl cold PBS. The cell suspension was then pelleted again at 500 g for 5 min at 4oC. For cell lysis, the pellets were resuspended in 50 µl cold ATAC-Resuspension Buffer (10 mM Tris-HCl pH 7.4 (Thermo Scientific, 10 mM NaCl (Sigma), and 3 mM MgCl2 (Sigma) in nuclease-free water (Thermo Scientific) containing 0.1% (v/v) NP40 (Sigma), 0.1% (v/v) Tween-20 (Sigma), and 0.01% (v/v) Digitonin (Promega) by gently pipetting up and down three times. After cell lysis, 1 ml of cold ATAC-Resuspension Buffer was added and mixed by inverting the tubes. The samples were then centrifuged at 500 g for 10 min at 4oC and supernatant is carefully removed away from the pellets. For the transposition reaction, each pellet was resuspended in 12.5 µl of transposition mix (6.25 µl 2x TD buffer (Illumina), 0.625 µl Tn5 (Illumina), 4.125 µl PBS, 0.125 µl 1% (v/v) Digitonin (Promega), 0.125 µl 10% (v/v) Tween-20 (Sigma), 1.25 µl nuclease-free water (Thermo Scientific) by gently pipetting up and down six times. The samples were then incubated at 37oC for 30 min in a thermomixer shaking at 300 rpm, followed by immediate purification using a DNA Clean & Concentrator-5 Kit (Zymo, Cat.# D4013) according to the manufacturer's specification, except for the final elution volume, which was 21 µl. A PCR reaction volume of 50 µl was then set up using 20 µl of the purified DNA, 25 µl of the NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs) and 1.25 mM of the ATACseq oligos published previously (Buenrostro et al., 2013). The DNA samples were amplified with the following parameters:  72oC for 5 min, 98oC for 30 s and 11 cycles of 98oC for 10 s, 63oC for 30 s and 72oC for 30 s. The omni-ATAC libraries were then purified using the DNA Clean & Concentrator-5 Kit (Zymo) according to the manufacturer's specification and eluted in 20 µl nuclease-free water (Thermo Scientific). The libraries were quantified with Qubit dsDNA HS Assay Kit (Invitrogen) and assessed for library size distribution using an Agilent Bioanalyzer.The libraries were then pooled and sequenced in 2x100 cycle paired-end mode on an Illumina Novaseq instrument.