GSM9633869: YO_GEX, scRNAseq; Mus musculus; RNA-Seq
Bone marrow cells were isolated from femurs, tibia, iliac crests, and spine of young and old mice. Red blood cells were removed by ACK lysis, and mononuclear cells were enriched by density gradient centrifugation. Hematopoietic stem and progenitor cells (HSPCs) were enriched using CD117 magnetic beads and sorted by flow cytometry as Lineage? Sca1+ c-Kit+ CD48? CD150+ cells. Single-cell libraries were generated using the 10x Genomics Chromium platform (GEM-X Single Cell 3? v4). Approximately 10,000?20,000 cells were captured per sample. Gene expression libraries were constructed following the manufacturer?s protocol. CellTag and hashtag oligo feature barcode libraries were captured simultaneously. Libraries were sequenced on an Illumina NovaSeq platform. Hashtag oligo-based multiplexing was performed using TotalSeq-B antibodies (BioLegend) following the 10x Genomics Feature Barcode protocol (3? Gene Expression with Antibody Capture). Cells from different biological samples were labeled with unique oligonucleotide-conjugated antibodies prior to pooling. The hashtag oligos (HTOs) enabled sample demultiplexing after sequencing. The following TotalSeq-B hashtag sequences were used: B0301 (ACCCACCAGTAAGAC), B0302 (GGTCGAGAGCATTCA), and B0303 (CTTGCCGCATGTCAT). Feature barcode libraries were generated alongside gene expression libraries and processed to assign cells to their sample of origin.