title: GSM3754902: Skin_Treg_4dup_2 [ATAC-seq]; Mus musculus; ATAC-seq
library_construction_protocol: Chromatin accessibility mapping on purified cell populations was performed using the ATAC-seq method as previously described (Buenrostro et al., 2013; Corces et al., 2017), with minor adaptations. Briefly, in each experiment 2,000 - 15,000 sorted cells were pelleted by centrifuging for 10 min at 4 °C at 500 x g. After centrifugation, the pellet was carefully lysed in 50 µl resuspension buffer supplemented with NP-40 (Sigma), Tween-20 and Digitonin (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1 % NP-40, 0.1 % Tween-20, 0.01 % Digitonin) and incubated for 3 minutes on ice. Then, 1 ml of ice-cold resuspension buffer supplemented with Tween-20 was added, and the sample was centrifuged at 4 °C at 500 x g for 10 minutes. The supernatant was discarded, and the cell pellet was carefully resuspended in the transposition reaction (25 µl 2 x TD buffer (Illumina), 2.5 µl TDE1 (Illumina), 16.5 µl PBS, 5 µl nuclease-free water, 0.5 µl 1% Digitonin (Promega), 0.5 µl 10% Tween-20 (Sigma)) for 30 min at 37 °C on a shaker at 1000 rpm. Following DNA purification with the Clean and Concentrator-5 kit (Zymo) eluting in 23 µl, 2 µl of the eluted DNA was used in a quantitative 10 µL PCR reaction (1.25 µM forward and reverse custom Nextera primers (Corces et al., 2017), 1x SYBR green final concentration) to estimate the optimum number of amplification cycles with the following program 72°C 5 min; 98°C 30 s; 25 cycles: 98°C 10 s, 63°C 30 s, 72°C 1 min; the final amplification of the library was carried out using the same PCR program and the number of cycles according to the Cq value of the qPCR. Library amplification using custom Nextera primers was followed by SPRI size selection with AmpureXP beads to exclude fragments larger than 1,200 bp. DNA concentration was measured with a Qubit fluorometer (Life Technologies). The libraries were sequenced using the Illumina HiSeq 4000 or NextSeq platforms.
attributes.tag: GEO Accession
attributes.value: GSM3754902