GSM2058240: Single cell 14 month old LMPP 19; Mus musculus; RNA-Seq
Bone marrow mononuclear cells (MNCs) from the iliac crest, femur, and tibial bones were isolated by Ficoll-Paque density gradient centrifugation (GE Healthcare). MNCs were depleted of cells expressing the mature lineage (Lin) markers CD3e, CD11b, CD45R/B220, Ly-6G/Ly-6C (Gr-1) and TER-119 using the IMag Mouse Hematopoietic Progenitor Cell Enrichment set (BD). The cells were then stained with combinations of anti c-Kit (clone 2B8), Sca-1 (clone E13-161.7), CD34 (clone RAM34), CD150 (clone mShad150), Flt3 (clone A2F10), mature Lin markers with fluorochrome-conjugated secondary antibody, and the viability dye propidium iodide (PI). LMPPs are gated as Lin- Sca-1+ c-Kit+ CD150- CD34+ Flt3hi. Isolated LMPPs from 4mo or 14mo mice were then resuspended at a concentration of 200 cells/μl and loaded onto the C1 Single-Cell Auto Prep Integrated Fluidic Circuit (Fluidigm) with freshly prepared staining buffer. Images of captured cells were collected with a Zeiss AxioObserver.Z1 microscope in the brightfield, GFP and CY3 channels using ZenPro software. Single-cell RNA extraction and mRNA amplification were performed following manufacturer’s recommendations including exogenous spike-in controls. library construction was performed following manufacturer’s recommendations including exogenous spike-in controls.
GEO Accession
GSM2058240