title: Primary human CD34+ cells for modeling ETO2-GLIS2 leukemogenesis - ATACseq
design: Primary human CD34+ cells for modeling ETO2-GLIS2 leukemogenesis - ATACseq
library_construction_protocol: 5x104-1x105 GFP+ live-sorted cells were spun at 500g for 5 min, washed with cold DPBS, lysed in cold lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.1% NP40, 3min on ice) and immediately spun at 500g for 10 min. The pelleted nuclei were resuspended in 50 µL of transposase reaction mix (Tagment DNA TDE1 Enzyme and Buffer Kit, Illumina) for 30 min at 39°C.  Transposed DNA was purified using a MinElute PCR purification kit  (Qiagen) in 15µL of nuclease-free H2O and amplified with NEBNext® Ultra™ II Q5® Master Mix and Nextera XT DNA Library Preparation Kit (Illumina), using the following PCR conditions: 72°C for 5 min, 98°C for 30s, then 12 cycles of 98°C for 10 s, 63°C for 30 s, and 72°C for 1 min.  Transposed DNA was purified using a MinElute PCR purification kit  (Qiagen) in 15µL of nuclease-free H2O and amplified with NEBNext® Ultra™ II Q5® Master Mix and Nextera XT DNA Library Preparation Kit (Illumina), using the following PCR conditions: 72°C for 5 min, 98°C for 30s, then 12 cycles of 98°C for 10 s, 63°C for 30 s, and 72°C for 1 min. Libraries were purified with AMPure XP beads (Beckman Coulter)