title: Illumina HiSeq 2500 paired end sequencing; Single-cell RNA-seq of Pdgfra+/Sca1+/Cd31- mouse cardiac cells
design: Single-cell RNA-seq of Pdgfra+/Sca1+/Cd31- mouse cardiac cells
library_construction_protocol: Pdgfra+ Sca1+ Cd31- cells were isolated using FACS sorting from ventricles of male adult mice. Briefly, hearts were minced and incubated in collagenase type II (Worthington, USA) at 37°C before to be filtered on 40μm strainers. Cells were then resuspended in red cell lysis buffer followed by dead cell removal and immunostaining for 15min on ice with fluorophore-conjugated antibodies and followed by FACS sorting for mentioned markers. Immediately after cell sorting and counting, the cells were loaded on the integrated fluidics circuits (IFC) C1 chip. A LIVE/DEAD cell staining kit (Life Technologies, USA) was used to mark live and dead cells with green (live) and red (dead) fluorescence before the run. Each capture site was carefully examined under a Leica fluorescence microscope in bright field, Red, and Green fluorescence channels for cell doublets and viability. Finally, 12 µl of cells (200,000 cells/ml) were re-suspended in 8 microliters of suspension reagent (Fluidigm), of which 7 microliters could be loaded on the Fluidigm Single Cell Auto Prep IFC optimized for 5-10 μm cell-size. The 96 trapping sites on the chip were visualized by the microscope to ensure the capture rate was satisfactory, before cell lysis and cDNA preparation. The reverse transcription was performed using Clontech SMARTer Ultra Low Input RNA Kit V4 (Takara-Clontech, USA) on the chip.  SMARTer V4  Nextera XT protocol from Illumina
attributes.tag: Experimental Factor: single cell identifier
attributes.value: SP_replicate2_Cell_097