GSM6323629: Tumor-bearing BM, scRNA-seq; Mus musculus; RNA-Seq
Spleens were minced and digested in 1mg/mL Collagenase Type IV + 0.25mg/mL DNase I. Bone marrow was removed by centrifugation and digested. Digestion was quenched then filtered through a 100μm filter. Cells were pelleted, counted, and aliquoted. TruStain FcX™ PLUS was used to block samples then biotin anti-lineage antibodies were used to stain lineage cells. After washing, strepavidin magnetic beads (NEB, S1420S) were used to deplete lineage positive cells. Remaining cells were pelleted and then stained with streptavidin BV605 (Biolegend, #405229), anti-CD45 AF700, anti-PDGFRα APC, anti-CD51 PE (Biolegend, clone RMV-7), anti-CD31 PE-Cy7 (Biolegend, clone 390), anti-Sca-1 PerCP-Cy5.5 (Biolegend, clone D7), and anti-c-Kit FITC (Biolegend, clone 2B8). Cells were then washed into holding buffer (0.04% BSA in PBS), stained with DAPI, and sorted on a high modified MoFlo into five populations: Live/Lin-/CD45+/c-Kit+/Sca-1+, Live/Lin-/CD45+/c-Kit+/Sca-1-, Live/Lin-/CD45-/CD31+, Live/Lin-/CD45-/CD31-, Live/Lin-/CD45-/CD31-/CD51+, Live/Lin-/CD45-/CD31-/CD51-. These populations were combined at equal ratios and submitted for 10X Genomics 3' v3.1 Chemistry sample preparation and sequencing on a NovaSeq6000. Library preparation was performed according to the manufacturer's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, cells 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 appropriately sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction an Ilumina R2 primer sequence was added.