Single-cell sequencing of individual murine aortal smooth muscle cells (C57BL/6)

ABSTRACT: Smooth muscle-specific EGFP reporter mice (Myh11-CreERT2pos;dTom/EGFP-reporter) were sacrificed and perfused with PBS. The aorta including common carotid arteries was dissected, minced, and enzymatically digested for 90-120 minutes while shaking at 37°C in a digestion mix containing collagenase II (2 mg/ml; Worthington), elastase-I (0.04 mg/ml; Sigma), and DNase1 (5 U/ml; New England Biolabs). Cell suspensions were serially filtered through 70µm and 40µm cell strainers followed by washing twice with PBS. Aortic smooth muscle cells were sorted on a JSAN cell sorter (Bay Biosciences, Japan) based on their EGFP expression.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. Raw data files were trimmed using Reaper with a quality cutoff of 53 (length 20 bp, prefix 50 bp) and a minimum clean read length of 50 bp. Reads were then mapped to the genome (mm10) using STAR. FeatureCounts was used to assign reads to genes from the Gencode vM6 annotation. Genes with less than 50 counts across all cells were discarded prior to normalization. The knn.error.models function from SCDE59 was used to construct cell-specific error models by evaluating the gene counts for a observed cell vs. the expected gene counts from the k most similar cells. The parameter k was chosen to resemble roughly three subpopulations. Estimated fragments per million mapped reads (FPM, in log scale) were then obtained using the scde.expression.magnitude function.

ORGANISM(S): Mus musculus

PROVIDER: GSE97955 | GEO | 2017/04/20

SECONDARY ACCESSION(S): PRJNA383478

REPOSITORIES: GEO