GSM9116219: GA_biol rep2; Homo sapiens; ATAC-seq
We spun 50,000 cells at 500 × g for 5 minutes, followed by a wash using 50 μL of cold 1x PBS and centrifugation at 500 × g for 5 minutes. Cells were lysed using cold lysis buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630). Immediately after lysis, nuclei were spun at 500 × g for 10 minutes using a refrigerated centrifuge. To avoid losing cells during the nuclei prep, we used a fixed angle centrifuge and carefully pipetted away from the pellet after centrifugations. Transpose and purify: Immediately following the nuclei prep, the pellet was resuspended in the transposase reaction mix (25 μL 2x TD buffer, 2.5 μL Transposase (Illumina) and 22.5 μL of nuclease free water). The transposition reaction was carried out for 30 minutes at 37 °C. Directly following transposition the sample was purified using a Qiagen Minelute kit Following purification, we amplified library fragments using 1x NEBnext PCR master mix and 1.25 μM of custom Nextera PCR primers 1 and 2 (see table below), using the following PCR conditions: 72°C for 5 minutes, 98°C for 30 seconds, followed by thermocycling at 98°C for 10 seconds, 63°C for 30 seconds and 72°C for 1 minute. To reduce GC and size bias in our PCR we monitored the PCR reaction using qPCR in order to stop amplification prior to saturation. To do this, we amplified the full libraries for 5 cycles, after 5 cycles we took an aliquot of the PCR reaction and added 10 μl of the PCR cocktail with Sybr Green at a final concentration of 0.6x. 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