Optimized ATAC-Seq Protocol for Adipocyte Nuclei

ATAC-Seq libraries were generated on 100,000 mature adipocyte nuclei using a modified protocol to that published recently20 (link). More precisely, transposase reaction was carried out for 30 min at 37 °C in a 25-μl reaction volume using 10X transposase concentration (Illumina Nextera Kit). EDTA (25 mM) was added to the reaction mix and transferred to ice before recovering DNA using MinElute PCR Purification columns (Qiagen). Next, samples were PCR enriched (ten cycles; Supplementary Table 6) and DNA was isolated using GeneRead Purification columns (Qiagen). Libraries were quantified by quantitative PCR (Supplementary Table 7), Picogreen and LabChip, then were sequenced on the Illumina HiSeq2500 pair-ended 100 bp, using the Nextera sequencing primers.
Raw reads were trimmed for quality (phred33 ≥30) and length (n≥32), and Illumina adapters were clipped off using Trimmomatic v. 0.22 (ref. 35 (link)). Filtered reads were aligned to the hg19 human reference using BWA v.0.6.1 (ref. 13 (link)). Peaks were called without a control using MACS v. 2.0.10.07132012 (ref. 36 (link)) at a q-value cutoff of 0.05.