GSM3772727: J1.DMSO_ATAC.Input_REP1; Mus musculus; ATAC-seq
Assay for Transposase Accessible Chromatin (ATAC)-seq experiments were carried out by adapting the protocol published by (Buenrostro et al., 2015). To allow for calibration of the ATAC-seq signal between different samples, 5 x 105 chicken DF-1 cells were spiked-in with 5 x 106 mouse ES cells from each experimental condition.  Cell mixtures were lysed in 1 ml of Lysis Buffer (50 mM KCl, 10 mM MgSO4.7H2O, 5 mM HEPES, 0.05% NP40 (IGEPAL CA630), 1 mM PMSF and 3 mM DTT) by incubating at room-temperature for 1 min followed by the two washes in 1 ml ice-cold RSB Buffer (10 mM NaCl, 10 mM Tris (pH 7.4), 3 mM MgCl2).   ATAC reactions were performed in 50 µl reactions in the presence of Tn5 reaction buffer (10 mM TAPS, 5 mM MgCl2 and 10% dimethylformamide), 5 x 104 nuclei (resuspended in H2O) and 2 µl of 12 µM Tn5 transposase (produced in house following a protocol by Picelli et al, 2014) for 30 min at 37 °C. DNA was purified using the QiaQuick MinElute columns (Qiagen) following manufacturer's instructions.   Input samples were prepared by subjecting genomic DNA that had been isolated from each sample prior to the ATAC reaction to Tn5 tagmentation in reactions containing 5 ng genomic DNA, 1 µl of Tn5 and incubated at 55 °C for 7 min. ATAC-seq libraries were prepared by PCR amplification using custom made Illumina barcodes in the NEBNext Ultra II Q5 2x PCR Master Mix with 6-12 cycles. PCR amplicons were purified and size selected using home-made SPRI beads (Rohland and Reich 2012) in two rounds (1:0.6 then 1:1.4 DNA:beads ratio). ATAC-seq libraries were quantified using the NEBNext Library Quant Kit and pooled prior to sequencing on an Illumina HiSeq4000 instrument to generate 75 bp paired-end reads.