GSM2264806: C15_4_1_ATAC-seq; Homo sapiens; OTHER
About 50,000 cells were collected and pelleted by centrifugation for 15 min at 500 g and 4°C. Cell pellets were washed once with ice-cold 1x PBS and then pelleted again by centrifugation at the previous settings. Cell pellets were re-suspended in 25 μl of ice-cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal CA-630), and nuclei were pelleted by centrifugation for 30 min at 500g, 4°C The nuclei were re-suspended in 50 μl reaction buffer (2 μl of Tn5 transposase and 12.5 μl of TD buffer from a Nextera DNA Library Prep Kit from Illumina, and 22.5 μl nuclease-free H2O). The reaction was incubated at 37°C for 30 min, and subsequently the reaction mixture was purified using MinElute PCR Purification Kit (Qiagen). The purified transposed DNA was amplified with NEBNext High-Fidelity 2 X PCR Master Mix (New England Biolabs) and custom-designed primers with barcodes (Buenrostro et al. 2013). Gel electrophoresis was used to remove primer dimers from the PCR products with 2% E-Gel EX Agarose Gels (Thermo Fisher Scientific), and then the PCR products were purified using QIAquick PCR Purification Kit (Qiagen). DNA concentration was measured with a Qubit Fluorometer (Thermo Fisher Scientific) and library sizes were determined using Agilent High Sensitivity DNA kit on Agilent 2100 Bioanalyzer. The ATAC-seq libraries were sequenced with a HiSeq 2000 sequencer (Illumina) with 2 X101 cycles.