title: GSM4257390: hESC rep2; Homo sapiens; ATAC-seq
library_construction_protocol: TruePrep DNA Library Prep Kit V2 Illumina (Vazyme, TD501 & TD202) The hESCs, 4i cells and EBs were washed once in PBS one time and dissociated into single cells with TrypLE Express enzyme or Trypsin. Then the dissociated cells were collected for next experiments. ATAC-seq was performed by using TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501&TD202) according to the manufacturer's recommendations. The details of the operation are listed. Different stages of cells according to human primordial germ cells like cells in vitro induction system were collected in PBS(2%BSA) and spun at 500 g at 4°C for 10 min. The pellet was resuspended in 50 μl lysis buffer and incubated at 4 °C for 15 min and then spun at 500 g at 4°C for 5 min. The supernatants were removed by carefully pipetting away from the pellets. For the transposition reaction, 10 μl 5X TTBL buffer, 5μl TTE Mix V50 were combined and added to each pellet up to 50 μl. The samples were incubatedat 37°C for 30 min followed by immediate purification using Beckman Beads according to the manufacturer's specification, except for the final elution volume, which was 24 μl. The PCR was set up in a 50 μl reaction volume using 24 μl of transposed DNA, 10 μl of 5x TAB, 5 μl PPM and 5 μl P5 and P7 primers TruePrepTM Index Kit V2 for Illumina® (Vazyme, TD202). PCR parameters were: 72 °C for 5 min, 98 °C for 30 s and 15 cycles of 98 °C for 10 s, 60 °C for 30 s and 72 °C for 30 s. The libraries were purified using a QIAGEN MinElute PCR purification kit (QIAGEN,Cat#28004) followed by Agencourt AMPure XP beads (Beckman Coulter, A63880) according to the manufacturer's recommendations. Library fragments ranging from 200 to 700 bp were enriched and the final elution volume was 30 μl. Libraries were sequenced pair-end 150bp sequencing on an Illumina HiseqXten at Novogene in Beijing (http://www.novogene.com/tech/service/seq/product/).
attributes.tag: GEO Accession
attributes.value: GSM4257390