|
| Status |
Public on Sep 01, 2020 |
| Title |
D06_1 |
| Sample type |
SRA |
| |
|
| Source name |
Retinal microvascular endothelial cells
|
| Organism |
Bos taurus |
| Characteristics |
passage: 3
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RMECs were isolated from bovine retinas according to (Stitt, Chakravarthy, Archer, & Gardiner, 1995). RMECs were maintained in DMEM supplemented with 0.1mg/ml Primocin, 0.38μg/ml insulin, 1mg/ml heparin and 10% (v/v) porcine serum (PS). BRECs were passaged at approximately 80% confluency in a 1:3 ratio and grown until confluency at passage 3 for Fluidigm C1 runs . RMECs were dissociated form flask using TryPLE Express dissociation reagent and single cells were captured on Fluidigm C1 Microfluidics chip.
Clontech modified Illumina® Nextera® XT DNA sample preparation protocol was used for single-cell mRNA sequencing library preparation from cDNA acquired using the C1 system which was miniaturised using the Labcyte Access Robotic System in conjunction with Echo 525 Acoustic Dispenser
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Fluidigm_1_expression_matrix.csv
Cell 20
|
| Data processing |
Base call files were generated on board the nextseq 500 sequencer using real time analysis (RTA) and then converted to fastq format post-run using bcl2fastq2 in BaseSpace which also removed illumina adapters
Sequencing QC was assessed using Fastqc (v0.11.3) to ensure good quality sequencing and that adapters were successfully removed
Sequenced reads (fastq format) were aligned to bovine genome UMD3.1 (Bos Tau4) using STAR (v2.5.3a) with default settings
Alignment QC was assessed using multiQC
Gene counts calculated using HTseq (v0.6.1p1) with default settings
Created expression matrix from HTseq output
Genome_build: UMD 3.1 (bos Tau4)
Supplementary_files_format_and_content: csv files with expression matrices created from HTseq output with genes in rows and single cells in columns
|
| |
|
| Submission date |
Sep 03, 2019 |
| Last update date |
Sep 01, 2020 |
| Contact name |
David Arthur Simpson |
| E-mail(s) |
david.simpson@qub.ac.uk
|
| Organization name |
Queen's University Belfast
|
| Department |
Wellcome-Wolfson Institite for Experimental Medicine
|
| Street address |
The Wellcome – Wolfson Institute for Experimental Medicine, 97 Lisburn Road
|
| City |
Belfast |
| State/province |
N. Ireland |
| ZIP/Postal code |
BT9 7BL |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL23055 |
| Series (1) |
| GSE136800 |
Novel insights into retinal microvascular endothelial cells revealed through single cell RNA-seq analysis. |
|
| Relations |
| BioSample |
SAMN12685241 |
| SRA |
SRX6796405 |
