ATAC-Seq was performed as previously described (Buenrostro et al., 2015 (link)), with some modifications. CD4 +T cells were activated with anti-CD3/CD28 in presence of IL-2 for 3 days. 50,000 cells were lysed and transpositions were performed using transposase mixture (Nextera DNA library prep kit, Illumina), supplemented with 0.01% digitonin (Promega; Madison, WI). Transposition reactions were incubated for 30 min at 37°C in a ThermoMixer (Eppendorf) with agitation at 300 rpm. DNA was purified using the MinElute Reaction Cleanup kit (Qiagen), and libraries amplified using NEBnext PCR master mix with the following primers:
Libraries were quantified using RT-qPCR prior to sequencing. All Fast-ATAC libraries were paired-end sequenced, 75 bp using a HiSeq2500 instrument. Quality of FASTQ files was performed using FASTX trimmer. More than 50 million reads were mapped, with <10% mapped, on average, to the mitochondrial genome. The reads were aligned to the hg19 (UCSC) version using Burrows-Wheeler Aligner (BWA-MEM). Peaks were called using MACS2 (Zhang et al., 2008 (link)) peak-caller, and the reads from input DNA sample were used as control. Visualization of the peaks was done using R Software.
Libraries were quantified using RT-qPCR prior to sequencing. All Fast-ATAC libraries were paired-end sequenced, 75 bp using a HiSeq2500 instrument. Quality of FASTQ files was performed using FASTX trimmer. More than 50 million reads were mapped, with <10% mapped, on average, to the mitochondrial genome. The reads were aligned to the hg19 (UCSC) version using Burrows-Wheeler Aligner (BWA-MEM). Peaks were called using MACS2 (Zhang et al., 2008 (link)) peak-caller, and the reads from input DNA sample were used as control. Visualization of the peaks was done using R Software.
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