GSM3406951: ATACseqUCLA1hESC3k; Homo sapiens; ATAC-seq
Cells were collected in lysis buffer (10mM Tris pH7.4, 10mM NaCl, 3mM MgCl2, 1% NP40) and spun at 500g for 10 min to collect nuclei for ATAC-seq library construction ATAC-seq was performed using Nextera DNA library prep kit (Illumina, 15028212) as previously described (Pastor et al., 2018). Cells were collected in lysis buffer (10mM Tris pH7.4, 10mM NaCl, 3mM MgCl2, 1% NP40) and spun at 500g for 10 min. The pellet was resuspended in the transposase reaction mix (25 μL 2× Tagmentation buffer, 2.5 μL transposase and 22.5 μL nuclease-free water) and incubated at 37°C for 30 min. The samples were purified using MinElute PCR Purification Kit (Qiagen, 28006) and amplified using 1× NEBnext PCR master mix (NEB, M0541S) and 1.25 μM of custom Nextera PCR primers 1 and 2 with the following PCR conditions: 72 °C for 5 min; 98 °C for 30 s; and thermocycling at 98 °C for 10 s, 63 °C for 30 s and 72 °C for 1 min. Samples were amplified for five cycles and an aliquot of the PCR reaction was used to determine the required cycles of amplification by real-time PCR. The remaining 45 μL reaction was amplified with the determined cycles and purified with MinElute PCR Purification Kit (Qiagen, 28006) yielding a final library concentration of about 30 nM in 20 μL. Libraries were subjected to single-end 50bp sequencing on HiSeq 2000 or HiSeq 2500 sequencer with 4-6 indexed libraries per lane.