title: GSM2913208: Rep2_vehicle_ATACSeq; Mus musculus; ATAC-seq
library_construction_protocol: ATAC-Seq was performed following a method published by Buenrostro et al. (Nat Methods. 2013, PMID: 24097267). Briefly, a homogenous single cell suspension was prepared by harvesting cells using a cell scraper and a cell strainer in cold PBS buffer. 50,000 cells were centrifuged at 500×g for 5 min, 4°C and washed with 50 μl of cold PBS buffer. The plasma membrane was lysed in 50 μl of cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40) and centrifuged immediately at 500×g for 10 min, 4°C to obtain nuclei. The nuclear pellets were reacted in the transposition reaction mixture of 1X Tagment DNA Buffer (FC-121-1030, Illumina) and Tn5 transposase (FC-121-1030, Illumina) for 30 min at 37°C. DNA was purified using a Qiagen MinElute PCR Purification Kit (QIAGEN). The transposed DNA fragments were amplified in the PCR mixture containing a specific index adapter (Illumina standard index adapter) for each sample by PCR. Amplified libraries were purified using a AMPure beads (1:1.8 ratio, BECKMAN COULTER). Libraries were quantified using a Qubit® dsDNA High-Sensitivity Assay Kit (Q32854, Life Technologies). Fragment size distribution was tested on E-Gel (2%, Invtrogen). DNA libraries were sequenced on an Illumina HiSeq 3000 platform (50 bp, Paired-end) by the NHLBI DNA Sequencing and Genomics Core, NHLBI.