|
| Status |
Public on Feb 13, 2012 |
| Title |
Retinal_microvascular_endothelial_cells |
| Sample type |
SRA |
| |
|
| Source name |
Retinal microvasculature
|
| Organism |
Bos taurus |
| Characteristics |
cell type: Primary retinal endothelial cells
passage: 2
|
| Growth protocol |
Retinal microvascular endothelial cells (RMECs) were isolated from bovine retina by established protocols. Briefly, bovine eyes were transported from a local abattoir on ice and the retinas removed and washed free of RPE in Dulbecco's minimal essential medium (DMEM; Life Technologies). The retina was cut into segments, homogenized in MEM and then filtered through an 85 µm filter. The trapped microvessels were digested at 37 °C for approximately 30 min in PBS containing 250 µg/ml pronase, 200 µg/ml DNAase, and 50 µg/ml collagenase. The filtrate was microscopically examined to determine the end point for maximum retrieval of endothelial cells. Vessel fragments were then trapped in a 53 µm filter which was centrifuged at 1100 rpm for 10 minutes. The pellet was resuspended in Dulbecco's modified Eagles medium (DMEM) containing antibiotic (0.1mg/ml Primocin), 0.38 µg/ml insulin, 1mg/ml heparin and 20% porcine serum (Sigma). This mixture was seeded into 25 cm2 gelatin-coated Falcon flasks and maintained at 37 °C in a mixture of 5% CO2 and air. 10% porcine serum was used in the culture medium from passage 1 onwards and cells were analysed at passage 2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using a microRNeasy kit (Qiagen). A small RNA library was prepared using a sample prep kit v1.5 kit (Illumina) following the manufacturer's protocol. Briefly, following linker ligation and PCR amplification the range of products corresponding to small RNAs were excised from a 6% polyacrylamide gel
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Data processing |
Adapter sequences were clipped from the raw sequences using Genomics workbench software (CLCbio, Aarhus, Denmark) and a list of unique sequences with the number of reads for each generated
|
| |
|
| Submission date |
Aug 11, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
David Arthur Simpson |
| E-mail(s) |
david.simpson@qub.ac.uk
|
| Organization name |
Queen's University Belfast
|
| Department |
Centre for Experimental Medicine
|
| Street address |
QUB Institue of clinical Science, Block A, RVH
|
| City |
Belfast |
| State/province |
N. Ireland |
| ZIP/Postal code |
BT12 6BA |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL11153 |
| Series (1) |
| GSE31340 |
Non-coding small RNA profiling by high throughput sequencing of bovine primary retinal microvascular endothelial cells |
|
| Relations |
| SRA |
SRX092596 |
| BioSample |
SAMN00710544 |
