GSM8101672: Patient SU302 Diagnosis timepoint Blast replicate 2 ATACseq Sample; Homo sapiens; ATAC-seq
Sorted AML blasts were prepared for ATAC-seq as previously described54. 5,000-50,000 cells were washed in cold FACS buffer and spun at 4°C for 5 minutes at 500 rcf in a fix-angled centrifuge. Cell pellets were then resuspended in 50 uL of ATAC-seq resuspension buffer (RSB: 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2 in water) with 0.1% NP40, 0.01% digitonin, and 0.1% Tween-20 and incubated on ice for three minutes. After lysis, 1 mL of ATAC-seq RSB with 0.1% Tween-20 was added and tubes were inverted six times to mix. Isolated nuclei were then spun at 4°C for 10 minutes at 500 rcf in a fix-angled centrifuge. Supernatant was removed and nuclei were resuspended in 50 uL transposition mix (25uL 2xTD buffer, 2.5 uL Tn5 transposase (100 nM final), 16.5 uL PBS, 0.5 uL 1% digitonin, 0.5 uL 10% Tween-20, and 5 uL nuclease-free water). Transposition reactions were incubated at 37°C for 30 minutes in a thermomixer with shaking at 1000 rpm. Reactions were cleaned up using Qiagen MinElute Reaction Cleanup kits and processed as previously described. All libraries were amplified with a target concentration of 20 μl at 4 nM, which is equivalent to 80 femtomoles of product. All libraries were sequenced on an Illumina NextSeq with 75 bp paired end reads.