|
| Status |
Public on Jun 18, 2019 |
| Title |
Heart, MI 250 (scRNA-seq) |
| Sample type |
SRA |
| |
|
| Source name |
cardiac endothelial cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: Pdgfb-iCreERT2-R26R-Brainbow2.1
developmental stage: adult
condition: 7-day post MI
tissue: heart
cell type: cardiac endothelial cells
|
| Treatment protocol |
iCreERT2 activity was induced using a single intraperitoneal injection of 150 mg/ kg tamoxifen in 200 μl peanut oil resulting in Brainbow2.1 transgene (RFP, nuclear GFP, YFP or membranous CFP) expression in Pdgfb-lineage ECs. At 14 days post-tamoxifen, myocardial infarction MI was induced by permanent ligation of the left anterior descending coronary artery in the MI group. At 7 days post-MI (21 days post-tamoxifen), hearts from the healthy control and MI groups were harvested into ice-cold sterile collection media. Ventricles were extracted, finely minced and digested to obtain single cell suspensions. Cells were FACS-sorted using antibodies against CD31 and Podoplanin, and CD31+Confetti+Podoplanin- cells were selected for further processing.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Single cell suspensions were processed using the 10X Genomics ChromiumTM Single Cell 3’ Assay, during which the cells were partitioned into Gel Bead-In-Emulsions (GEMs) and polyadenylated (polyA) transcripts were captured.
Cells were incubated in GEMs with primers containing an Illumina R1 primer sequence, a 16 bp 10X barcode, a 10 bp Unique Molecular Identifier (UMI) and a poly-dT primer sequence and full-length cDNAs were generated from the captured polyA transcripts. cDNAs were then amplified by PCR followed by enzymatic fragmentation and size selection for optimised amplicons. P5, P7, i7 sample index and R2 primer sequence were added during library construction via end-repair, A-tailing, adaptor ligation and PCR.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
*I1_001.fastq.gz: contains the 8 nt sample index.
*R1_001.fastq.gz: contains the cell barcode (first 16 nt) and UMI (next 10 nt)
*R2_001.fastq.gz: contains the 75 nt RNA sequence.
Raw reads were aligned to the mouse reference genome mm10 (Ensembl 84) using the 10X Genomics Cell Ranger Single Cell 2.1.0 pipeline.
The output filtered feature-barcode matrix (containing detected cellular barcodes only) together with the corresponding feature and barcode sequences files for each sample from the Cell Ranger pipeline were read into R (v3.5.2) using the Seurat (v2.3) package, which returned a unique molecular identified (UMI) count matrix. The matrices from the eight samples were used to create the corresponding objects and then aggregated to generate a single Seurat object with the raw count matrix stored. The aggregated raw count matrix is provided here as the processed data with the column names as the cell names and row names as the feature/gene names.
Genome_build: mm10 (Ensembl 84)
Supplementary_files_format_and_content: Aggregated_raw_read_counts.txt
|
| |
|
| Submission date |
Jun 17, 2019 |
| Last update date |
Jun 19, 2019 |
| Contact name |
Mairi Brittan |
| E-mail(s) |
mbrittan@ed.ac.uk
|
| Organization name |
University of Edinburgh
|
| Department |
Centre for Cardiovascular Science
|
| Street address |
47 Little France Crescent
|
| City |
Edinburgh |
| ZIP/Postal code |
EH16 4TJ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL21103 |
| Series (1) |
| GSE132880 |
Single cell RNA-seq of adult mouse heart endothelial transcriptomes following myocardial infarction |
|
| Relations |
| BioSample |
SAMN12082862 |
| SRA |
SRX6081262 |
