GSM6208872: ATAC-seq_R2_HOT_minus; Mus musculus; ATAC-seq
iMEF cells were treated with HOT or vehicle for 7 days. Adherent cells were washed with PBS twice, trypsinized and collected in a 15 ml falcon. Cell count were measured using a Innovatis CASY Cell counter. 10^5 cells were transferred in 1.5 ml reaction tube. The cells were centrifuged at 500 x g for 5 min at 4°C and carefully re-suspended in ice-cold 1xPBS. Cell count were measured another time with Innovatis CASY cell counter. 5*10^4 cells per sample were aliquoted in fresh tubes. Cells were spin down at 500 x g for 5 min at 4°C and subsequently the supernatant removed. For ATAC-Seq basically the protocol of Corces et al., Nature Methods 14, 959-962 (2017) with adaptions of Thomas Look & Prof. Martin Zenke (Institute of Biomedical Engineering, Cell Biology, Aachen University Medical School, Germany can be found in Li et al., BMC Genome Biology 20, 45 (2019)) was employed.  Crude nulcei extracts were prepared by adding 50 µl of ice-cold ATAC-RSB (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) supplemented with 0.1 % NP40, 0.1% Tween-20, 0.01% Digitonin. Cells were incubated for 3 mins on ice. Cooled 1ml Wash buffer (ATAC-RSB + 0.1% Tween-20) was added, tubes inverted and centrifuged for at 500 x g for 10 min at 4°C afterwards. With the pellets of the crude nuclei extracts, transposition procedure was performed. 50 µl Transposition mix (1xTD buffer, Tagment DNA Enzyme TDE1 both part of the Nextera Rapid Capture Exome Kit #FC140-1000 by Illumina, PBS, 0.1% Tween-20, 0.01% Digitonin) was added to nuclei samples and incubated for 1h at 37°C while shaking in a thermoblock. Purification of transposed DNA was done with the Quiagen MinElute PCR Purification Kit (#28004).  Transposed DNA was barcoded using i5/i7 primers of the Nextera Rapid Capture Custom Enrichment Kit (FC-140-1007 by Illumina) and NEBNext Ultra II Q5 Master Mix (#M0544) by New England Labs. Amplification with barcoding primers was done for initial 5 cycles. After this an aliquote of PCR product was taken and supplemented with SYBR Green in DMSO (SYBR Gold Nucleic Acid Gel Stain #S11494, Thermo Fisher) together with the attributed set of primers per sample and fresh Q5 polymerase. Another 20 cycles on this aliquot were performed and the amplification was monitored in a real-time application (RotorGeneQ, Qiagen) to assess the progress of the library preparation after the initial 5 cycles in the thermocycler. Both +HOT treated and one –HOT treated sample 4 additional cycles and for the second –HOT replicate 3 additional amplification cycles were needed to perform to extend the initial 5 cycles of preparation. After library preparation was done, samples were purified with the Quiagen MinElute PCR Purification Kit (#28004). The libraries were prepared using the Next Ultra II kit (New England BioLabs) and the sequencing was performed as pared-end reads for 75 cycles with a NextSeq 500/550 High Output kit v2 (Illumina) according to the manufacturer's recommendations.