GSM8147284: Sen1_ATAC; Mus musculus; ATAC-seq
1. Prepare nuclei. To prepare nuclei, we spun 50,000 cells at 500g for 5 min, which was followed by a wash using 50 µL of cold 1× PBS and centrifugation at 500g for 5 min. Cells were lysed using cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630). Immediately after lysis, nuclei were spun at 500g for 10 min using a refrigerated centrifuge. To avoid losing cells during the nuclei prep, we used a fixedangle centrifuge and carefully pipetted away from the pellet after centrifugations. 2. Transpose and purify. Immediately following the nuclei prep, the pellet was resuspended in the transposase reaction mix (25 µL 2× TD buffer, 2.5 µL transposase (Illumina) and 22.5 µL nucleasefree water). The transposition reaction was carried out for 30 min at 37 °C. Directly following transposition the sample was purified using a Qiagen MinElute kit.3. PCR. Following purification, we amplified library fragments using 1× NEBnext PCR master mix and 1.25 µM of custom Nextera PCR primers 1 and 2 (Supplementary Table 1), using the following PCR conditions: 72 °C for 5 min; 98 °C for 30 s; and thermocycling at 98 °C for 10 s, 63 °C for 30 s and 72 °C for 1 min. To reduce GC and size bias in our PCR, we monitored the PCR reaction using qPCR in order to stop amplification before saturation. To do this, we amplified the full libraries for five cycles, after which we took an aliquot of the PCR reaction and added 10 µl of the PCR cocktail with Sybr Green at a final concentration of 0.6×. We ran this reaction for 20 cycles to determine the additional number of cycles needed for the remaining 45-µL reaction. The libraries were purified using a Qiagen PCR cleanup kit yielding a final library concentration of ~30 nM in 20 µL. Libraries were amplified for a total of 10–12 cycles The libraries were subsequently sequenced on an Illumina NovaSeq 6000 sequencing machine, producing paired end reads with a length of 150 base pairs (bp).