Cultured human umbilical cord derived CD34+ human hematopoietic stem and progenitor cells (HSPCs)
tissue
Cultured human umbilical cord derived CD34+ human hematopoietic stem and progenitor cells (HSPCs)
Stage
BFUE
surface markers to isolate cells by facs-based methods
IL-3R−CD34+CD36−
Sequenced DNA Library
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
other
library_construction_protocol
Progenitor cells at each stage were sorted by a combination of cell surface markers for IL-3R, CD34 and CD36. Erythroblasts at each stage of terminal erythroid differentiation were sorted by a combination of cell surface markers for glycophorin A, band 3, and α4 integrin. Cells were lysed in buffer (10mM Tris-HCl, 10mM NaCl 3mM MgCL2 and 0.1% IGEPAL) for 15 minutes at 4C, then centrifuged and re-suspended with transposase reaction mix (2x tagmented DNA buffer, transposase [Ilumina Nextera]), and nuclease-free water) and incubated for 30 min at 37 degrees. DNA was purified using a MinElute column (Qiagen) and PCR amplified in KAPA HiFi 2x mix (Kapa Biosystems) with barcoding primers (Buenrostro et al., 2013) at 98C for 45 sec (45s) + 5 x at (98C for 15s + 63C for 30s + 72C for 30s) + 72C for 1 min. PCR products were cleaned with MinElute columns and size exclusion was performed with magJet NGS Cleanup and Size Selection kit (Thermo Fisher). Q-PCR library amplification test and PCR library amplification was performed as previously described(Buenrostro et al., 2015).