GSM5058791: MiaPaCa2_ATAC_rep1; Homo sapiens; ATAC-seq
50000 cells were pelleted by centrifugation and re-suspended in 50 µl of cold lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% NP40, 0.1% Tween-20 and 0.01% Digitonin). After incubation on ice for three minutes, lysis was washed out by adding 1 mL of lysis buffer without NP40 and digitonin. Nuclei were pelleted by centrifugation for 10 min at 500g, 4 ºC.  The supernatant was discarded and nuclei were re-suspended in 50 µl of reaction buffer containing 1 µl of Tn5 transposase (made in house),  10 ul of 5x transposase buffer (50mM Tris-HCl, pH 8.4 and 25mM MgCl2), 16.5 µl PBS, 0.5 µl of 1% digitonin, 0.5 µl of 10% Tween-20. The reaction was incubated at 37ºC for 30 minutes with 600 RPM mixing. Then 5 µl of clean up buffer (900mM NaCl, 300mM EDTA), 2ul of 5% SDS and 2 µl of Proteinase K (20 µg/µl) (New England Biolabs) were added and incubated for 30 min at 40 ºC. Tagmented DNA was isolated using SPRI beads (2x) and amplified by PCR. Fragments smaller than 600 bp were isolated using SPRI cleanup and libraries where sequenced on an Illumina NextSeq500 platform.