Nextera tn5 transposase

For RT-qPCR, total RNA was extracted and purified using the Quick-RNA MiniPrep Kit (Zymo Research). The quantity and quality of RNA was determined using a spectrophotometer. RNA was reverse transcribed using the High-Capacity RNA to cDNA Kit (Applied Biosystems) according to the manufacturer’s instructions. For ATAC-qPCR, cells were processed as described above for ATAC-Seq. For qPCR, nuclei were isolated from 50,000 cells and sequencing adapters were transposed for 30 min at 37 °C using 2.5 μl of TDE1 (Nextera Tn5 transposase, Illumina). The reaction was terminated and purified using MinElute reaction cleanup (Qiagen) and used as template. In both cases, qPCR was performed using 1 μl of 5 μM Powerup SYBR green PCR Master Mix (Applied Biosystems), 2 μl of dH₂O and 2 μL of cDNA sample in a 10 μl reaction. Each measurement was carried out in a duplicate using a CFX384 Real-Time System (Bio-Rad). The PCR conditions were: 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 60 s. For ATAC-qPCR, primers were designed to flank the center of the DAR as determined by ATAC-Seq. Primer sequences used are listed in Supplementary Table 2. Gene expression levels were normalized to β-actin, and accessibility changes were normalized to GAPDH.

ATAC-seq was performed with two independent experiments per condition^{60 (link)}. Briefly, nuclei were isolated from 50,000 sorted cells per replicate using a solution of 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl₂, and 0.1% IGEPAL CA-630. Immediately following nuclei isolation, the transposition reaction was conducted using Nextera Tn5 transposase and TD buffer (Illumina) for 45 min at 37 °C. Transposed DNA fragments were purified using a Qiagen MinElute Kit, barcoded with dual indexes (Illumina Nextera), and PCR amplified using NEBNext High Fidelity 2 × PCR master mix (New England Labs). The size distribution and molarity of the sequencing library were determined by using an Agilent Bioanalyzer and KAPA quantitative RT-PCR (KAPA Biosystems). Sequencing was performed using an Illumina HiSeq X ten platform to acquire at least ∼ 50 M fragments per sample. Paired-end reads were mapped to the hg38 reference genome using Bowtie2. Only concordantly mapped pairs were kept for further analysis. Peak calling was performed using MACS v1.4 to identify areas of sequence tag enrichment. These peaks were displayed in the IGV genome browser and further processed for annotation and for differential open chromatin detection.