|
| Status |
Public on Sep 19, 2018 |
| Title |
DT ex vivo cell DT_D2_105 |
| Sample type |
SRA |
| |
|
| Source name |
Medial layer of mouse aorta
|
| Organism |
Mus musculus |
| Characteristics |
region: Descending thoracic aorta
genotype: wild type
cell type: Vascular smooth muscle cell
background: C57Bl/6
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Aortas from healthy male mice (8-14 weeks) 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, AA and DT segments isolated and 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. The medial layer from 5-8 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. The AA and DT samples were processed using a Fluidigm C1 system. Cell suspensions (100 cells/μl) were loaded onto medium-sized (17-25 μm) Auto Prep Arrays (Fluidigm) and processed according to the manufacturer’s instructions. The loaded arrays were visually assessed under an inverted microscope to select capture sites containing a single cell, yielding a 40-70% capture efficiency.
Cells were processed using the SMARTer® Ultra™ Low RNA Kit (Clontech) and amplified cDNA was isolated from the arrays. Sequencing libraries were made from 0.125-0.375 ng amplified cDNA using the Nextera XT library preparation kit (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Single-cell RNA seq, Fluidigm C1
normalised_aadt_counts.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
|
| Street address |
Francis Crick Avenue, Cambridge Biomedical Campus
|
| City |
Cambridge |
| State/province |
Cambs |
| ZIP/Postal code |
CB2 0QH |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE117963 |
Disease-relevant transcriptional signatures identified in individual smooth muscle cells from healthy mouse vessels |
|
| Relations |
| BioSample |
SAMN09745588 |
| SRA |
SRX4493261 |
