Single-cell RNA sequencing facilitates quantitative analysis of mouse hematopoietic stem/progenitor cells and SCF/2i expanded cells

Purpose: The goal of this study is to compare the transcriptome profiling of SCF/2i expanded cells to freshly sorted hemtopoietic stem/progenitor cells (HSPCs) at single cell level. Methods: Mouse bone marrow HSPCs (HSC, MEP, CLP, CMP, GMP) and alive SCF/2i expanded cells were sorted by FACS. The FACS-sorted single cell suspensions were washed with ice cold 0.04% BSA in PBS, then loaded onto 3' library chips as per the manufacturer's protocol for the Chromium Single Cell 3' Library (10X Genomics, V2). The 10× Genomics libraries were first sequenced on an Illumina NextSeq 500 aiming at a coverage of 5,000 raw reads per cell to estimate the cell numbers, before being sequenced deeper on an Illumina HiSeq 2500 aiming at a coverage of 50,000 raw reads per cell. (paired-end; read1: 26 cycles; i7 index: 8 cycles; read 2: 98 cycles). Raw sequencing data were pre-processed using the Cell Ranger pipeline (10× Genomics, v 2.1.0). The raw-count datasets from difference cell types were integrated and analyzed by Seurat3 R package. Results: tSNE plot showed that the transcriptome of SCF/2i cells overlapped with that of freshly sorted GMPs. The top 50 highly expressed genes from each comparison were used for analysis by heatmap, which showed that the SCF/2i cells shared a similar gene expression pattern with GMPs, but not other hematopoietic cell lineages. Conclusion: The SCF/2i expanded cells are GMPs. Overall design: Comparation of the transcriptome between SCF/2i expanded cells and HSPCs