title: GSM7965636: GMP replicate3, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: The femur, tibia, and fibula of eight to twelve-week-old Balb/C mice were dissected, carefully removing muscle and tendons. The bones were then crushed in PBS containing 0.2% BSA at pH 7.4 using a mortar and pestle. The cell suspension was passed through a 40-μm nylon mesh sieve and then lysed to remove red blood cells with red blood cell lysis buffer.  To enrich hematopoietic progenitors, the bone marrow cells were incubated with an APC-conjugated anti-c-Kit antibody (ACK2, eBioscience), washed and incubated with anti-APC microbeads (Miltenyi Biotech, Germany). After incubation, the cells were washed and re-suspended in MACS buffer and separated through a magnetic column on a magnetic separation plane (Miltenyi Biotech). Cells remained on the column were collected. For live cell analysis or sorting, the collected cells were first stained with biotin-conjugated anti-mouse lineage cocktail antibodies [Ter-119 (TER-119), Gr-1 (RB6-8C5), CD11b (M1/70), B220 (RA3-6B2), CD4 (RM4-5) and CD8 (53-6.7), Biolegend]. After washing, the cells were stained with streptavidin-conjugated fluorescein (Biolegend) and the following antibodies protected from light: c-Kit (ACK2, eBioscience), Sca-1 (El3161.7, Biolegend), CD34 (RAM34, BD), Flt3 (A2F10, BD), CD16/32 (93, BD). DAPI (Solarbio) was used to exclude dead cells. The cells were washed between each step. FACS sorting was performed using a BD LSRII sorter.  The isolated progenitor populations were defined as follows:  LMPP, Lin-c-Kit+Sca-1+CD34+Flt3+; CMP, Lin-c-Kit+Sca1-CD34+CD16/32low; GMP, Lin-c-Kit+Sca-1-CD34+CD16/32high. The Libraries were constructed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, samples were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction.