Comparative Transcriptomic Analysis of Hematopoietic System Across Species by Microwell-Seq. Comparative Transcriptomic Analysis of Hematopoietic System Across Species by Microwell-Seq

Traditional single-cell RNA-seq technologies have shown limitations for large scale experiments. Here, we constructed a single-cell resolution transcriptomic atlas of (HSPCs) in human and mouse,including a total number of 71,729 single cells clustered into 39 subpopulations by computational analysis. In human, we found 20 progenitors primed with unique lineages like megakaryocyte, neutrophil and B cell progenitor can project directly from HSPCs. Within the c-Kit positive cells from mouse bone marrow, we described 19 progenitor subgroups at single cell resolution. Overall design: We sampled 7 batches (CD34+_b1-7) of Human CD34+ mobilized peripheral blood stem cells (PBSCs); 2 batches (CD34+_thawed_l6-7) of thawed Human CD34+ mobilized peripheral blood stem cells (PBSCs) and 2 batches (CD34-_b1-2) of thawed Human CD34- mobilized peripheral blood stem cells (PBSCs), around 5000 single cells per sample. 1 batch of mouse-transferred CD34+ cells; 3 batches of Mouse c-kit+ cells and 5 batches of mouse bone marrow cells.1 batch of zebrafish kidney marrow-derived hematopoietic cell. Human CD34+ cells were selected by human CD34 selection kit (StemCell Technologies Cat #18056), following the protocol provided by the manufacturer. After spin at 300 x g for 5min, the supernatant was removed and CD34+ cells were suspended in 1x DPBS + 2mM EDTA and diluted to ~ 100,000/mL for Microwell-seq. After thawing in IMDM medium, human CD34+ cells were suspended in 100μL of PBS+5% FBS for exposure of antibodies. mPB CD34+ cells were transplanted into sublethally irradiated NCG (NOD-Prkdcem26Cd52Il2rgem26Cd22/Nju, Nanjing Biomedical Research Institute of Nanjing University) mice via an injection into the femur cavity (approximately, 1X105 cells per mouse). Two months after transplantation, hCD45+CD34+ cells (mouse-transferred CD34+ cells) were sorted from mice bone marrow cells collected from the femur and tibia. Mouse c-kit + cells were were sorted by flow cytometry, following the protocol provided by the manufacturer. The following antibodies were used for MPP(CD34+CD38-Thy1-CD45RA-CD49f-) cell sorting: CD34 PE (Biolegend, clone 581, cat #343506), CD34 FITC (BD bioscience, clone 581, cat #555821), CD38 PE-Cy7 (BD bioscience, clone HIT2, cat #560677), CD90 APC (BD bioscience, clone 5E10, cat #559869), CD45RA APC/Fire 750 (Biolegend, clone HI100, cat #304151), CD49f BV421 (Biolegend, clone GoH3, cat #313623).