title: GSM2583704: Single-cell sequencing of individual murine aortal smooth muscle cells [Harmandeep_SMC_run2_55]; Mus musculus; RNA-Seq
library_construction_protocol: Single-cell transcriptome analysis was performed on a C1 Autoprep station (Fluidigm) using 5-10µm mRNA Arrays and standard protocols. 15.000 events sorted on a JSAN swift  sorter were suspended in 10 µl resuspension buffer (PBS + 0.5% bovine serum albumin + 2nM EDTA, sterile filtered) and further diluted (6:4) in suspension buffer. 3µl were loaded to the C1 Array while remaining cells (~10.000) were used for total RNA isolation using µRNeasy micro kit (Qiagen) combined with on-column DNase digest (Qiagen). RNA was quantified by Qubit HS RNA Assay (Thermo Fisher) and used for tube control. Pre-amplification and cDNA synthesis were done with Smarter ultra-low RNA Kit (Clontech) without changes to the standard protocol. cDNA obtained from C1 system was quantified using PicoGreen assay (Thermo Fisher) and diluted to 0.1–0.3 ng/μl. Picking of positive cDNA samples and library preparation were done with NGS Express pipetting robot (Perkin Elmer) using custom made protocols. Barcoded and amplified libraries were pooled in groups of 16 libraries for final SRRI-bead based cleanup and quantified by Qubit HS dsDNA Assay (Thermo Fisher) and BioAnalyzer 2100 HS DNA Assay (Agilent). Sequencing was performed on Nextseq500 Sequencer (Illumina) using v2 chemistry and High Output Flow Cell with 75bp single end protocol.
attributes.tag: GEO Accession
attributes.value: GSM2583704