GSM5655509: AU-CBH-15330 @1uM for 1h-R1; Homo sapiens; ATAC-seq
Nuclei extraction and tagmentation of open chromatin was performed as described in the Buenrostro et al. 2013 Nat Methods. ATAC-seq was performed as previously described in Buenrostro et al. 2013 Nat Methods. In brief, 50,000 cancer cells were washed in cold PBS and resuspended in cytoplasmic lysis buffer (CER-I from the NE-PER kit, Invitrogen, cat. no. 78833). This single-cell suspension was incubated on ice for 5-8 min (depending on the cell line) with gentle mixing by pipetting every 2 min. The lysate was centrifuged at 1,300g for 5 min at 4 °C. Nuclei were resuspended in 50μl of 1× TD buffer, then incubated with 2-2.5 μl Tn5 enzyme for 30 min at 37 °C (Nextera DNA Library Preparation Kit; cat. no. FC-121-1031). Samples were immediately purified by Qiagen minElute column and PCR-amplified with the NEBNext High-Fidelity 2X PCR Master Mix (NEB; cat. no. M0541L) following the original protocol27. qPCR was used to determine the optimal PCR cycles to prevent over-amplification. The amplified library was further purified by Qiagen minElute column and SPRI beads (Beckman Coulter; cat. no. A63881). ATAC-seq libraries were sequenced on the Illumina HiSeq 2500 (125-nucleotide read length).