title: GSM4879720: WT mESCs - ATAC-seq; Mus musculus; ATAC-seq
library_construction_protocol: 50,000 FACS isolated cells were collected at 500 x g for 5 min at 4 degrees celsius and then washed in 50 ml of cold PBS. Cells were collected at 500 x g for 5 min at 4_x0004_C and the cell pellets were resuspended in 50 ml of cold ATAC lysis buffer (10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630, 10 mMTris pH 7.4) by pipetting slowly up and down. The samples were spun down immediately at 500 x g for 10 min at 4 degrees celsius  and the supernatants were removed by carefully pipetting away from the pellets. For the transposition reaction, 22.5 ml of UltraPure Distilled Water (Life Technologies, Cat#10977-015), 25 ml of Tagment DNA Buffer (Illumina, Cat#15027866) and 2.5 ml of Tagment DNA Enzyme 1 (Illumina, Cat#15027865) were combined and added to each pellet. The samples were then incubated at 37 degrees celsius for 30 min followed by immediate purification using a MinElute Reaction Cleanup Kit (QIAGEN, Cat#28204) according to the manufacturer's specification, except for the final elution volume, which was 11 ml. The PCR was set up in a 50 ml reaction volume using 11 ml of transposed DNA, 25 ml of the NEBNext High-Fidelity 2x PCR Master Mix (Cat#M0541S) and 1.25 mM of the ATACseq oligos published previously (Buenrostro et al., 2013). PCR parameters were: 72 degrees celsius for 5 min, 98 degrees celsius for 30 s and 9 cycles of 98 degrees celsius for 10 s, 63 degrees celsius for 30 s and 72 degrees celsius for 1 min. Libraries were purified using a QIAGEN MinElute PCR purification kit (QIAGEN, Cat#28004) followed by Agencourt AMPure XP beads (Beckman Coulter, Cat#A63880) according to the manufacturer's recommendations. For the library construction, library fragments ranging from 150-600bp were enriched and libraries were sequenced in 2x75 cycle paired-end read format, with a total of 150.8 million reads, on a NextSeq550 High output v2.5 Illumina machine.