|
| Status |
Public on Jun 22, 2023 |
| Title |
HFF ABRAXAS2 transfected CGH bulk |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
ABRAXAS2 CGH bulk
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HFF
|
| Treatment protocol |
HFF cells were transfected with 16.9 μg Cas9 RNP and 5 μM of Alt-R® Cas9 Electroporation Enhancer by electroporation using the AMAXA™ 4D-499 Nucleofector™ device (Lonza®, Bale, Switzerland) with P3 Primary Cell Line and CZ-167
|
| Growth protocol |
HFF cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM with glucose (4.5 g.L-473 1), L-Glutamine (1 g.L-1) and pyruvate supplemented with 20% fetal bovine serum, MEM non-essential 474 amino acids 100X (Gibco® by ThermoFisher scientific, Carlsabad, CA, USA), 100 U/mL penicillin, and 475 100μg/mL streptomycin (all from Eurobio, Courtaboeuf, France)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from cultivated cells using the Wizard manual kit (Promega Corporation, Madison, USA).
|
| Label |
Cy5
|
| Label protocol |
DNA (minimum 125ng) was labeled (cyanine 3 or cyanine 5) using the Genomic DNA ULS Labeling Kit from Agilent Technologies (Agilent Technologies, Santa Clara, USA)
|
| |
|
| Channel 2 |
| Source name |
Control HFF WT
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HFF
|
| Treatment protocol |
HFF cells were transfected with 16.9 μg Cas9 RNP and 5 μM of Alt-R® Cas9 Electroporation Enhancer by electroporation using the AMAXA™ 4D-499 Nucleofector™ device (Lonza®, Bale, Switzerland) with P3 Primary Cell Line and CZ-167
|
| Growth protocol |
HFF cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM with glucose (4.5 g.L-473 1), L-Glutamine (1 g.L-1) and pyruvate supplemented with 20% fetal bovine serum, MEM non-essential 474 amino acids 100X (Gibco® by ThermoFisher scientific, Carlsabad, CA, USA), 100 U/mL penicillin, and 475 100μg/mL streptomycin (all from Eurobio, Courtaboeuf, France)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from cultivated cells using the Wizard manual kit (Promega Corporation, Madison, USA).
|
| Label |
Cy3
|
| Label protocol |
DNA (minimum 125ng) was labeled (cyanine 3 or cyanine 5) using the Genomic DNA ULS Labeling Kit from Agilent Technologies (Agilent Technologies, Santa Clara, USA)
|
| |
|
| |
| Hybridization protocol |
Labeled DNAs were hybridized onto Agilent oligonucleotide microarrays (8x60K Agilent Genetisure or 180K CGH + SNP) according to the manufacturer’s instructions (Agilent Technologies)
|
| Scan protocol |
Scanning of the microarrays was performed using a G5761A scanner (Agilent Technologies).
|
| Description |
Test 1
|
| Data processing |
Data analysis was carried out with Agilent Technologies software, namely Feature Extraction for Cytogenomics Algorithm v5.0 for the fluorescence ratio calculation and Agilent CytoGenomics v5.2 for the localization of chromosomal imbalances and LOH.
Deletions and duplications in the heterozygous state were characterized by values of the log2 ratio of fluorescence intensities (cyanine5/cyanine3) below -0.5 and above +0.3, respectively, with the statistical algorithm ADM2 used at a threshold of five. LOH was evaluated with the statistical algorithm ADM2 used at a threshold of 6 (Default Analysis Method v2)
|
| |
|
| Submission date |
Jun 15, 2023 |
| Last update date |
Jun 22, 2023 |
| Contact name |
Julian Boutin |
| E-mail(s) |
julian.boutin@u-bordeaux.fr
|
| Organization name |
INSERM U1312 BRIC
|
| Lab |
BioGO team 8
|
| Street address |
146 rue Leo Saignat
|
| City |
Bordeaux |
| State/province |
Nouvelle Aquitaine |
| ZIP/Postal code |
33000 |
| Country |
France |
| |
|
| Platform ID |
GPL17052 |
| Series (1) |
| GSE235019 |
Human HFF cells large rearranements detection after CRISPR-Cas9 nuclease (Fig.2c) |
|
