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Sample GSM3316059

Query DataSets for GSM3316059

Status

Public on Sep 19, 2018

Title

Lineage positive Sca1 negative cell CpSn.sca1_18_f

Sample type

SRA

Source name

Medial layer of mouse aorta

Organism

Mus musculus

Characteristics

region: Aorta
genotype: Myh11-CreERt2/Rosa26-Confetti
cell type: Vascular cell
background: C57Bl/6

Extracted molecule

total RNA

Extraction protocol

Myh11-CreERt2/Rosa26-Confetti males received intraperitoneal injections of tamoxifen in corn oil, at 6-8 weeks of age (10 injections of 1 mg/ml each) for lineage labelling. Aortas of healthy male mice were dissected free of fat and connective tissue. To generate single-cell suspensions, endothelial cells were removed manually from dissected aortas using a cotton bud and vessels incubated for 10 minutes in Collagenase Type IV (1 mg/ml, Life Technologies) and porcine pancreatic elastase (1 U/ml, Worthington BioChem) to allow for separation of the adventitia and medial cell layers. Tissue isolated from 5-11 animals was pooled and further digested for 1-2 hours to achieve a single-cell suspension, which was filtered through a 40 μm cell strainer. Individual Sca1-positive (S+) and Sca1-negative (S-) cells expressing a single VSMC-lineage marker (L+) or no VSMC-lineage marker (L-) were FACS-sorted on an Aria-Fusion flow cytometer (BD Bioscience) into separate wells of a 96-well plate. Sca1+ and Sca1- cells were included on the same plate and processed together to generate amplified cDNA using the Smart-seq2 protocol carried out as described previously (Picelli et al, Nature protocols 2014), with the following minor modifications: Primescript (Clontech) was used for reverse transcription, 24 PCR cycles were used for amplification of cDNA, and ERCC control RNA (Invitrogen) was added (1:40,000,000 or 1:80,000,000 dilution in RT-mix).
After quality assessment by Bioanalyzer (Agilent) and Quant-iT PicoGreen (Thermo Fisher) quantification, sequencing libraries were prepared from amplified cDNA (approximately 1-6 ng per sample) using the Nextera XT library prep kit (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Single-cell RNA seq, Smart-Seq2
normalised_counts_smartseq2_sca1.txt

Data processing

Samples 1 - 313: Raw reads were trimmed using TrimGalore and Cutadapt. Alignment of the reads to the GRCm38 genome was performed using TopHat. Reads per gene were counted using htseq-count (single-cell RNA-seq) or Seqmonk (bulk RNA-seq). Reads were normalised using DESeq2 (bulk RNA-seq) or the scran Bioconductor R package (sinlge-cell RNA-seq) using the computeSumFactors function.
Samples 314 - 317: Raw sequencing data were processed through the 10X genomics Cellranger pipeline (v2). Cellranger demultiplexed the raw sequencing data, produced the fastq files, aligned the reads to the mouse genome (mm10, STAR aligner) performed unique molecular identifier (UMI) counting.
Genome_build: mm10
Supplementary_files_format_and_content: Samples 1 - 313: Tab separated text files contain normalised read counts with genes in rows and cells/samples in columns. Samples 314 - 317: Filtered gene barcode matrices output by the 10X genomics Cellranger pipeline in the h5 format (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/advanced/h5_matrices)

Submission date

Jul 31, 2018

Last update date

Sep 19, 2018

Contact name

Steven William Wingett

E-mail(s)

steven.wingett@mrc-lmb.cam.ac.uk

Organization name

MRC Laboratory of Molecular Biology

Department

Cell Biology