title: GSM3763732: 24 Y Female run2518_lane12_read1 Cell_28; Homo sapiens; RNA-Seq
library_construction_protocol: Fluidigm C1 protocol Nextera XT DNA Library Preparation Kit (Illumina Inc, Santa Clara, CA) All aged (65-75 yo) samples were derived from femurs from individuals undergoing hip replacement surgery at the University of Michigan hospital. Donors had no known history of hematological malignancy. Bone marrow mononuclear cells (BM MNC) were isolated using ficoll based density centrifugation and cryopreserved. Additional bone marrow mononuclear cells were purchased from Allcells, Hemacare, Stemcell Technologies, University of Pennsylvania Stem Cell and Xenograft Core, and Cincinnati Children's Hospital Processing Core for young (18-30 yo) and middle-aged (45-55 yo) donors. For scRNA-seq, bone marrow cells from healthy donors was obtained at Cincinnati Children's Hospital Medical Center through informed consent under an approved institutional review board research protocol. Mononuclear cells were isolated with Ficoll-Paque (GE Healthcare), then enriched using CD34 MicroBeads on a Automacs Pro separator (Miltenyi, San Diego, CA). CD34+ cells were stained with lineage antibodies conjugated to PerCP-Cy™5.5: CD2 (Becton, Dickinson and Company, clone RPA-2.10), CD3 (Becton, Dickinson and Company, clone UCHT1), CD4 (Becton, Dickinson and Company, clone RPA-T4), CD7 (Becton, Dickinson and Company, clone M-T701), CD8 (Becton, Dickinson and Company, clone RPA-T8), CD10 (Becton, Dickinson and Company, clone HI10a), CD11b (Biolegend, clone ICRF44), CD14 (Becton, Dickinson and Company, clone M5E2), CD19 (eBioscience, clone HIB19), CD20 (eBioscience, clone 2H7), CD24 (Biolegend, clone ML5), CD56 (Becton, Dickinson and Company, clone B159), CD66b (Becton, Dickinson and Company, clone G10F5), Glycophorin A (Biolegend, clone HIR2) and CD90-biotin (eBioscience, clone 5E10). To isolate CD34+CD38- hematopoietic stem and progenitor cells (HSPC), lineage stained cells were stained with: Streptavidin APC-Cy7 (Becton, Dickinson and Company), CD123-PE (eBioscience, clone 6H6), CD34-APC (eBioscience, clone 4H11), CD38-PE-Cy7 (eBioscience, clone HIT2), CD45RA-FITC ( Invitrogen, clone MEM-56). Cell sorting was performed on a BD FACSAria II with a 100um nozzle. cDNA was made with Clontech SMARTer® Kit “the mRNA Seq: RT + Amp (1771x), Libraries were constructed using Nextera XT DNA Library Preparation Kit Single cells were separated using Fluidigm C1 system