Single-cell RNA-seq reveals differentiation of bona fide human pDCs and cDC1s in cultures of cord blood CD34+ progenitors, and a newly identified terminal differentiation step of cDC1s

CD34+ cord blood hematopoietic progenitors were expanded in vitro as previously described (Balan et al., J Immunol, 2014) and then differentiated on a mixed feeder layer of OP9 cells expressing or not the Notch ligand Delta-like 1, with FLT3-L, TPO and IL-7. At the end of the cultures, single live Lin- HLA-DR+ cells were index sorted in 96-well plates containing lysis buffer, and snap frozen. Four putative cell types were sorted according to their expression patterns of key combinations of cell surface markers: putative pDCs, putative cDC1s, putative pre-cDC2s and putative cDC2s. Single cell RNA-sequencing libraries were subsequently generated for 90 single cells and 6 control wells using an adaptation of Smart-Seq2 (Villani et al., Science, 2017). Cells were sequenced at a depth of 1-3M reads/cell. A total of 90 single cells and 6 controls from one culture were processed using a protocol adapted from Smart-Seq2 protocol (Villani et al., Science, 2017), which allows for the generation of full-length single cell cDNA, and sequencing libraries were generated using Illumina Nextera XT DNA library preparation kit. A few samples (10) were profiled but excluded from the processed data since they were either bulk (5) or blank (1) control samples or excluded due to QC (4). Therefore, there are 86 samples included here.