GSM2695950
GSM2695950: Aged_1_C52; Mus musculus; RNA-Seq
Single long-term hematopoietuc stem cells (LT-HSC: Lineage-, Sca1+, cKit+, Flt3-, CD48-, CD150+) were captured on a 5–10 μm cell diameter integrated microfluidic chip (IFC) using the Fluidigm C1 Single-cell AutoPrep system (Fluidigm Corporation). Cells were pre-stained with Calcien AM/EthD-1 LIVE/DEAD cell viability assay (Life Technologies) and loaded onto the IFC at a concentration of 500–700 cells/ul.  Viable single cell confirmation was performed with phase-contrast fluorescence microscopy to assess the number and viability of cells per capture site. Only single, live cells were included in the analysis. For RNA-seq analysis, cDNAs were prepared “on-IFC” using the SMARTer Ultra Low RNA kit for Illumina (Clontech) following fluidigm recomemdnations. Single-cell cDNA size distribution and concentration was assessed with PicoGreen (Life Technologies) and Agilent Bioanalyzer 2100 analysis (Agilent Technologies). Illumina libraries were constructed in 96-well plates using Illumina’s NexteraXT DNA Sample Preparation kit following the protocol supplied by Fluidigm. For each C1 experiment, a bulk RNA control and a negative control were processed in parallel, using the same reagent mixes as used on chip. Libraries were quantified by Agilent Bioanalyzer, using High Sensitivity DNA analysis kit, and also fluorometrically, using Qubit dsDNA HS Assay kits and a Qubit 2.0 Fluorometer (Technologies). Fluidigm C1 full-length single-cell RNAseq
GEO
GSM2695950
GEO Accession
GSM2695950