GSM4506443: H9ESC_PM [ATAC-seq]; Homo sapiens; ATAC-seq
The ATAC-Seq samples were prepared following the Omni-ATAC Seq protocol (Corces et al., 2017). In brief, single cell suspension of PM cells and BA cells were generated using Accumax and collagenase Mix as described in the cell culture and differentiation session of method. 50,000 cells were pelleted at 500 RCF at 4 °C for 5 mins. After removal of supernatant, pellet was resuspended in sterile water-based resuspension buffer containing 10 mM Tris-HCl pH=7.4 (Thermo Fisher, 15567-027), 10 mM NaCl (Thermo Fisher, AM9760G), 3 mM MgCl2 (Thermo Fisher, AM9530G), 0.1% NP-40 (Abcam, ab142247), 0.1% Tween-20 and 0.01% Digitonin (Thermo Fisher, BN2006) and incubated on ice for 3 minutes. Subsequent to incubation, lysis was washed by a water-based washing buffer containing 10 mM Tris-HCl pH=7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% Tween-20. The lysis was then subject to centrifuge at 500 RCF at 4 °C for 5 mins. After removal of supernatant, pellet was resuspended in 50 μl of transposition mixture consisting of 25 μl 2X TD Buffer (illumina, 15027866), 2.5 μl Tn5 transposase (illumina, 15027865), 16.5 μl PBS, 0.5 μl 1% Digitonin, 0.5 μl 10% Tween-20, 5 μl H2O and incubated at 37 °C for 30 minutes. The reaction mix was later cleaned up using Zymo DNA Clean and Concentrator-5 kit (Zymo Research, D4014). Library was then constructed by PCR amplification as instructed in the Omin-ATAC protocol and purified by Omega Mag-Bind RXNPure plus beads (Omega, M1386-01). Single-end libraries were construced and then sequenced in NextSeq platform with read length 75 bp. Each sample has more than 30 millions read per end.
GEO Accession
GSM4506443