GSM6616796: WT-STAT5-KLS; Mus musculus; RNA-Seq
Bone marrow cells were harvested from the femurs and tibias of 4 Vav1-Cre/+ or Vav1-Cre/+/STAT5abflox/flox (8-12 weeks). The cells were lineage depleted using a mouse lineage cell depletion kit (Miltenyi Biotech, Auburn, CA) and stained with FITC-labeled lineage antibodies (Gr-1, Mac-1, B220, Ter119, CD3, CD4, CD8a), anti-c-Kit-APC, anti-Sca-1-PE-Cy7 (Thermo Fisher Scientific, Norcross, GA), and dead cells were excluded by staining with propidium iodide dye. KLS cells were sorted using BD FACSAria III Cell Sorter. For sorting bone marrow stromal cells, cells were isolated as described [reference: Houlihan, D.D.; Mabuchi, Y.; Morikawa, S.; Niibe, K.; Araki, D.; Suzuki, S.; Okano, H.; Matsuzaki, Y. Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-alpha. Nature protocols 2012, 7, 2103-2111, doi:10.1038/nprot.2012.125]. Briefly, mouse bones (tibia and femur) were isolated and cleaned from 6-8 weeks old mice, then were crushed and cut into tiny pieces with sterile scissors, incubated them in 20 ml of preheated DMEM plus 0.2% (wt/vol; 40 mg) collagenase at 110 rpm at 37°C for 1 hour ). The cell suspension was filtered through a 70-μm cell strainer and bone fragment was further crushed and washed to liberate cells out of the bone into solution. CD45+ cells were depleted with mouse CD45 microbeads from Miltenyi Biotech( Auburn, CA) and stained with anti-Ter119-PE  (BD Biosciences, San Jose, CA), anti-CD45-FITC, Anti-CD71-Alexa 700, Anti-Sca-1-PE-Cy7 and Anti-CD140a-APC (Thermo Fisher Scientific, Norcross, GA). CD45negTer119-/lowCD71-Sca-1+CD140a+  cells were sorted using BD FACSAria III Cell Sorter. Sorted cells were immediately processed for single cells RNA sequencing. Tissue derived single cells were loaded onto the 10X Chromium Controller targeting 3000-6000 cells/sample. Single cell capture, barcoding, GEM-RT, clean-up, cDNA amplification and library construction were performed according to the manufacturers' instructions for v3 chemistry using 10x Genomics Single Cell 5' Chromium Chip B kit (PN-1000074) and Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3 (PN 1000092).  Final gene expression library pools were sent to Genewiz (South Plainfield, NJ) and sequenced on an Illumina's Novaseq6000 instrument with a targeted sequencing depth of 150,000 reads/cell.