GSM5234441: ATAC-seq_H1hESC_control_biologicalRep2; Homo sapiens; ATAC-seq
NET-CAGE: Nascent RNA from flash-frozen cells was isolated as described by Hirabayashi et al. 2019 Nat. Genet. with the following exceptions: (i) 5× DNase I enzyme (Thermo Fisher Scientific) was used to prepare the DNase I solution (50 μl), (ii) the samples were incubated for up to 1 h at 37°C while being pipetted up and down several times every 10 min, and (iii) RNA quality was measured using a TapeStation 4200 (Agilent). The ATAC-sequencing libraries were prepared as in Buenrostro et al 2015 Curr. Protoc. Mol. Biol. 5×104 EmGFP (-) and EmGFP (+) TetOn-hESCs (H1 clone 2, H1 clone 8, H9 clone 3 and H9 clone 4) were centrifuged at 500×g for 5 min. The pellets were washed in cold 1× PBS by centrifugation at 500×g for 5min. Each cell pellet was lysed in 50 μl of cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.1% IGEPAL CA-630) and centrifuged at 500×g at 4°C for 10 min. The pellet was then resuspended in the transposase reaction mix (2.5 μl of transposase in TD buffer (Nextera DNA library preparation kit, Illumina) and incubated at 37°C for 30 min. The reactions were purified through columns and eluted in 20 μl. After addition of the barcode oligos the DNA samples were amplified for 12 cycles (98°C for 10 s, 63°C for 30 s and 72°C for 60 s) in Phusion PCR master mix (Thermo Fisher Scientific). The PCR products were purified through the columns and eluted in 20 μl. NET-CAGE: CAGE-based libraries were generated according to the no-amplification non-tagging CAGE libraries for Illumina next-generation sequencers (nAnT-iCAGE) protocol (Murata et al 2014; Methods Mol. Biol.). All CAGE-based libraries were sequenced in single-read mode on an Illumina NextSeq500 platform.