GSM9308533: Old_mHSC_Ex_Vivo; Mus musculus; RNA-Seq
Bone marrow (BM) cells were flushed out of the femur and tibia bones in ice-cold IMDM and 2% FBS. Flow cytometry and FACS (fluorescence-activated cell sorting) were performed as previously described (Rimmele et al., 2015; Yalcin et al., 2010). In brief, total BM cells were immune-stained with fluorochrome-labeled antibodies for surface markers, such as lineage markers (CD5, B220, CD11b, Gr-1, 7-4, and Ter-119) using a lineage cocktail consisted of biotinylated hematopoietic multilineage monoclonal antibodies (StemCell Technology), CD45.1, CD45.2, stem and progenitor markers (c-Kit, Sca-1, CD48, CD150) for 15 min. followed by incubation with APC/CY7–streptavidin to identify progenitors (Lin- Sca1- c-Kit+) and LT-HSCs (LSK CD48- CD150+).  All samples were stained with DAPI for live cells. FACS isolation was performed using FACS Aria-II. Freshly sorted, viable HSCs (12000 -16000) from pooled young or old mice, or old HSCs cultured with ConA (40 nM), ddC (40 µM), or DMSO for 16 hrs were washed and re-suspended in 50µl 0.04% BSA/PBS, and proceeded to single cell RNA-Seq on 10X genomics platform from Genomics Core Facility at the Icahn School of Medicine at Mount Sinai. Briefly, single cells were partitioned, barcoded, and Gel Beads-in-emulsion (GEM) were generated using Chromium Next GEM Chip J Single Cell kit. Libraries were then prepared using Chromium Next GEM Single Cell 3′ Kits v3.1 (10x Genomics) and were sequenced using NovaSeq S4 200 cycle v1.5.