|
| Status |
Public on Jun 04, 2015 |
| Title |
Fibroblast_GM969_scrambled_shXPA_replicate-1 |
| Sample type |
RNA |
| |
|
| Source name |
Fibroblast_GM969_scrambled_shXPA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Human primary fibroblast GM969
treatment: Human primary fibroblast GM969 transfected with a scrambled shXPA
|
| Treatment protocol |
The shRNA vector against XPA was from Sigma-Aldrich. Lentiviral production was performed by co-transfection of packing plasmid pCMV-dr8.2 DVPR (Addgene plasmid 8455) , envelope vector pCMV-VSV-G (Addgene 8454), and the abovementioned XPA shRNA plasmid or a scrambled plasmid (Addgene 1864) into 293T cells using X-tremeGENE transfection reagents (Roche). For infection, the virus was incubated with GM969 primary fibroblast cells plus 4 µg/ml Polybrene for 2 days followed by 24 ~ 48 h 2ug/ml puromycin selection, cells were then applied for further experiments.
|
| Growth protocol |
XPA-deficient (GM04312C) and the matched complemented (GM15876A) cells, were maintained in Gibco DMEM medium supplemented with 10% FBS with 1% penicillin-streptomycin (P&S). Primary fibroblasts, GM969, were cultured in Gibco MEM medium mixed with 15% FBS, 1% P&S, and 1 Х Glutamine. All cells were grown in 20% O2/5% CO2 at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction was done using a TRIzol Plus RNA purification kit as per manufacturer’s protocol. Quality and quantity of the total RNA was tested using the Agilent 2100 Bio-Analyzer and RNA 6000 nano kits.
|
| Label |
Streptavidin-Cy3 bound to biotin labeled cRNA.
|
| Label protocol |
Standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) In short, 0.5ug of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.
|
| |
|
| Hybridization protocol |
Standard Illumina protocol. In short, a total of 0.75ug of biotin-labeled cRNA was hybridized at 58 degrees C for 16 hours to Illumina's SentrixHumanHT-12 v4 Expression BeadChips (Illumina, San Diego, CA). Each array on the HumanHT-12 v4 Expression BeadChip targets more than 47,000 probes derived from the National Center for Biotechnology Information Reference Sequence (NCBI) RefSeq Release 38 (November 7, 2009) and other sources and offers genome-wide transcriptional coverage of well-characterized genes, gene candidates, and splice variants, with approximately 15-fold redundancy. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.
|
| Scan protocol |
Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina.
|
| Description |
Fibroblast_GM969_scrambled_shXPA_replicate-1
|
| Data processing |
Data was extracted using the Illumina GenomeStudio software(v1.9.0). Any spots at or below the background were filtered out using an Illumina detection p value of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below. Z-score = (raw value - avg)/std.
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| |
|
| Submission date |
Mar 01, 2014 |
| Last update date |
Jun 22, 2020 |
| Contact name |
Supriyo De |
| Organization name |
NIA-IRP, NIH
|
| Department |
Laboratory of Genetics and Genomics
|
| Lab |
Computational Biology & Genomics Core
|
| Street address |
251 Bayview Blvd
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21224 |
| Country |
USA |
| |
|
| Platform ID |
GPL10558 |
| Series (2) |
| GSE55484 |
Defective Mitophagy in XPA via PARP1 activation and NAD+/SIRT1-depletion: Implications for neurodegeneration (human) |
| GSE55486 |
Defective Mitophagy in XPA via PARP1 activation and NAD+/SIRT1-depletion: Implications for neurodegeneration |
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