GSM9154744: T-47D cells, DMSO, 24 hr, biological replicate 3; Homo sapiens; ATAC-seq
We washed the cells three times with ice-cold PBS, then incubated them with 0.25% Trypsin-EDTA for 5 minutes. We quenched the trypsin with complete media (twice the volume) and centrifuged the cells at 500 × g for 5 minutes. Cells were washed twice more with ice-cold PBS, resuspended in ATAC resuspension buffer (RSP; 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2), and counted. We aliquoted 50,000 cells per biological replicate, centrifuged them, and resuspended the pellets in lysis buffer (RSP supplemented with 0.1% Tween-20, 0.1% NP-40, and 0.01% Digitonin) for a 5-minute incubation. We added 1 mL of wash buffer (RSP with 0.1% Tween-20), centrifuged the cells again, and resuspended them in tagmentation buffer (Diagenode 2× buffer [C01019043] diluted to 1×, supplemented with 0.01% Digitonin, 0.1% Tween-20, and 2.5 µL Diagenode Tn5 enzyme [C01070010]). We incubated the reaction for 30 minutes at 37°C. We purified the DNA using the Zymo DNA Clean & Concentrator kit, following the manufacturer's instructions, and eluted in 10 µL. For library amplification, we used the NEBNext® High-Fidelity 2X PCR Master Mix (M0541) and Diagenode UDI Tagmentation Library Set (C01011034) following this thermal cycling protocol: 72°C for 5 minutes, 98°C for 30 seconds, 10 cycles of: 98°C for 10 seconds, 63°C for 30 seconds, and one last cycle of 72°C for 1 minute. We size-selected libraries within a range of 200-700 bp using SPRISelect (B23317) beads, pooled the libraries, and sequenced them using a NextSeq 1000 platform.