Tagment dna buffer from nextera dna sample prep kit

1 × 10⁴ mTECs or fibroblasts from 4–6-week-old female Adig mice were used for preparation of ATAC-seq libraries, adapting published protocols^{23 (link),57 (link)}. Briefly, cells were suspended in 100μl of cold hypotonic lysis buffer [10mM Tris-HCl (pH 7.5), 10mM NaCl, 3mM MgCl2 and 0.1% NP40], followed by immediate centrifugation at 550g for 30 min. The pellet was re-suspended in 5μl of transposition reaction mix [1μl of Tagment DNA Enzyme and 2.5μL of Tagment DNA Buffer from Nextera DNA Sample Prep Kit (Illumina), 1.5μl H₂O], and was incubated for 60 min at 37°C for DNA to be fragmented and tagged. For library preparation, two sequential 7-cycles of PCR were performed in order to enrich small tagmented DNA fragments. After the first PCR, the libraries were selected for small fragments (less than 600bp) using SPRI beads followed by a second round of PCR with the same conditions in order to obtain the final library. Libraries were sequenced on the NextSeq 500 instrument to generate paired-end short reads (50bp, forward; 34bp, reverse). Data were processed essentially as per ChIP-seq analysis, except reads mapping to mitochondrial DNA (1~7%) were removed before analysis and peaks were identified using the ‘factor’ parameter in the findPeaks command of the Homer package.