title: GSM4332507: GFP4_II; Mus musculus; RNA-Seq
library_construction_protocol: Bone marrow from the tibia, femur, pelvic bones, sternum, and spine was harvested by crushing the bones with a mortar and pestle in a PBS buffer supplemented with 5% new born calf serum. Cells were triturated to obtain a single cell suspension, and bone debris was removed by filtering through a 70μm cell strainer. Red cells were lysed with an ammonium chloride lysis buffer. Cells were first stained with biotin-conjugated lineage marker's anti-CD48, and anti-Flk2, followed by anti-biotin superparamagnetic beads (Dynabeads® Biotin Binder, Life technologies), and lineage marker expressing cells were depleted via magnetic separation. The fraction enriched for Lineage-/low cells was further incubated with antibodies against CD150, c-Kit, Sca1, and fluorophore-conjugated streptavidin. HSCs were sorted as LSK/CD48-/Flk2-/CD150+ cells, and the HSC population were sorted into subpopulations based on label retention of H2BGFP. Cells were sorted into PBS with 5% newborn calf serum, pelleted, and resuspended in Trizol for RNA extraction. Glycoblue (15ug/ml) was added prior to RNA precipitation. Libraries were prepared according to manufactures instructions (Clontech, SMART-Seq v4 Ultra Low Input RNA Kit). Briefly, up to 1ng of Total RNA from each sample was taken for library cDNA synthesis. The resulting cDNA was quantified using Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen), and thecDNA integrity was assessed by Bioanalyzer.  100-300pg of cDNA was used for further library preparation uwing Nextera XT DNA Sample Preparation Kit (Illumina). Barcoded library concentrations and quality were assessed by Qbit (Invitrogen) and Bioanalyzer (Agilent).
identifiers.namespace: GEO
identifiers.id: GSM4332507