GSM4083935: Runx1cNeg_0Dox ATAC-Seq; Homo sapiens; ATAC-seq
50,000 single-cell sorted hematopoietic progenitors were pelleted and snap-frozen upon resuspension in 5 μl sucrose freezing buffer, consisting of 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 10 mM Tris pH 7.4 and 1.5 M sucrose. Transposition reaction was performed for 30 minutes at 37°C upon addition of 45 μl of ATAC reaction mix consisting of 25 μl Tagmentation DNA Buffer (Illumina cat# FC-121-1030, Nextera DNA Library Prep Kit), 2.5 μl Tn5 Transposase enzyme (Illumina cat# FC-121-1030, Nextera DNA Library Prep Kit), 1 μl of 0.5% Digitonin (Promega cat# G9441) and 16.5 μl water. DNA was purified using a MinElute Reaction Cleanup Kit (Qiagen cat# 28204). Purified DNA fragments were amplified using Customized Nextera PCR Primer Adaptors and NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs cat# M0541). Adaptor dimers were cleaned up from the libraries using AMPure magnetic beads (Beckman Coulter) prior to validation. Libraries were evaluated using a High Sensitivity DNA chip on an Agilent Technologies 2100 Bioanalyser™ instrument and concentration was measured using a RT-qPCR-based method (KAPA Library Quantification Kit for Illumina Sequencing Platforms). Libraries were also validated by RT-qPCR evaluation of the ratio of open (TBP promoter) to closed regions of DNA (chromosome 18) and active gene body (β-actin).