GSM8790748: FYT scRNAseq; Mus musculus; RNA-Seq
Tibiae and femora were harvested and cleaned of muscle and tissue. The bone marrow was isolated at 2500 g for 40 seconds, then resuspended into 100 μl of PBS, and kept on ice. Red blood cells were lysed using RBC lysing buffer for 5 minutes at room temperature, followed by a PBS wash. To enrich MkP cells, BM cells were stained with CD41+ and CD42d+ antibodies and sorted using MACS, followed by a BD FACSAria III 4L. Cells from 12 young mice were pooled in two tube (6 male, 6 females), and the cells from 11 old mice were pooled into another (5 males, 6 females). The pooled cells were then counted and resuspended at a concentration of 1000 cells/μL. Each cell was individually captured and uniquely barcoded using the 10X Chromium, following the manufacturer's protocol (10XGenomics, CA, USA). After generating Gel Beads-in-emulsion (GEMs), cell lysis and Gel bead dissolution within each reaction vesicle allowed for reverse transcription of polyadenylated mRNA, resulting in cDNA tagged with both a universal cell barcode and a unique molecular index (UMI). The cDNA library was then amplified and sequenced using the Illumina NovaSeq 6000 platform (Novogene, Singapore).