|
| Status |
Public on Sep 08, 2021 |
| Title |
Commercial_hCMECs_line_knock_out_RNF213_confluency_001_clone2_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Commercial human cerebral microvascular endothelial cells line
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: cerebral microvascular endothelial cells
transfection: Knock out RNF213 (Cas9 with guide RNA)
culture: Confluent cells
age: Adult
Sex: F
|
| Treatment protocol |
we generated RNF213 hCMECs knockout cells using CRISPR/Cas9 genome editing technology. Two sgRNAs were designed and cloned into expressing vector. csgRNAa cleavage efficiencies were calculated using mismatch assays (sgRNA-A, 41%; sgRNA-B, 40%). Both sgRNAs were cloned in a home-made lentiviral vector co-expressing the SpCas9and a puromycin N-acetyltransferase gene conferring resistance to puromycin antibiotics exposure. A negligible amount of RNF213 protein expression was observed in hCMECs RNF213-/- cells. Gene expression profiling analysis also revealed that transduced hCMECs preserved key endothelial markers. The sgRNAs used to produce the invalidation of RNF213 were specific for their designed RNF213 target and that the effects of knockout were not the result of unintended genomic alteration.
|
| Growth protocol |
hCMECs were purchased from Cedarlane Laboratories and were maintained in a gelatin-coated tissue culture flask in EBM-2 basal medium supplemented with 5% foetal bovine serum , 1.4 mM hydrocortisone, 5 mg/ml ascorbic acid, 1% chemically defined lipid concentrate, 1 ng/ml human basic fibroblast growth factor, 10 mM HEPES and the antibiotics, 100 IU/ml penicillin G and 25 μg/ml gentamicin and grown in an incubator at 37°C, 8% CO2 and 95% relative humidity. The culture medium was changed 3 times a week
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Cat#74104)
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng RNA using the One-Color LowInput QuickAmp Labeling Kit One-Color (Agilent) according to the manufacturer's instructions
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >8.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole SurePrint G3 Human GE 8x60K (G4851A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent SureScanner (G4900DA) using one color scan setting for 1x60k array slides (Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression of hCMECs line at confluency knock out for RNF213 in passage 33
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10,7 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Sep 07, 2021 |
| Last update date |
Sep 08, 2021 |
| Contact name |
Gaëtan Le-Bel |
| E-mail(s) |
gaetan.lebel17@gmail.com
|
| Organization name |
CUO-Recherche
|
| Lab |
Sylvain Guérin
|
| Street address |
Local H2-00, Hôpital du Saint-Sacrement,1050 Chemin Sainte-Foy
|
| City |
Québec |
| State/province |
Quebec |
| ZIP/Postal code |
G1S 4L8 |
| Country |
Canada |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE183603 |
Gene expression in commercial human cerebral microvascular endothelial cells (hCMECs) line knock out or not for RNF213 |
|
