|
| Status |
Public on Sep 30, 2020 |
| Title |
C1ag3257-3: Fibroblast_Ctr for HGPS affected-1_rep.3 |
| Sample type |
RNA |
| |
|
| Source name |
Skin fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
gender: Male
disease status: Clinically unaffected
age (years): 36
condition: PDL 12
condition: Untransformed
tissue: Skin fibroblasts
|
| Treatment protocol |
2x106 cells were seeded in 10 cm plates and grown overnight, the cells were then rinsed in cold PBS prior to RNA extraction.
|
| Growth protocol |
Dermal fibroblasts from HGPS patients and clinically unaffected parental controls were obtained from the Coriell Institute for Medical Research. Fibroblast cells were grown in Dulbecco modified eagle medium (DMEM), supplemented with 15% fetal bovine serum (FBS) (Gibco BRL), 50 µg/ml streptomycin and 50 units/ml penicillin, at 37oC with 5% CO2. HGPS patient AG03198 (10 yo female) was compared to AG03258 control (36 yo male, father); HGPS patient AG03513 (13 yo male) was compared to AG03512 control (41 yo female, mother); and HGPS patient AG6917 (3 yo male) was compared to AG6299 control (34 yo female, mother).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Trizol (Invitrogen) according to the manufacturer’s instructions. RNA quantity and integrity was evaluated by Nanodrop and Agilent Bioanalyzer RNA 6000 Chip (Agilent, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) In short, 0.5 μg 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 0.75 μg of biotin-labeled cRNA was hybridized at 58°C for 16 hours to Illumina Human HT12v4 Expression Bead Chips (Illumina, San Diego, CA). The arrays were then rinsed, blocked and the biotin-labeled cRNA was detected by staining with streptavidin-conjugated Cy3.
|
| Scan protocol |
Arrays were scanned at a resolution of 0.54 μm using the Illumina iScan scanner.
|
| Data processing |
Data was extracted using the Illumina GenomeStudio software V2011.1, Gene expression module 1.9.0. Any spots at or below the background were filtered out using an Illumina detection Pvalue 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. Complete data including detection scores is supplied.
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| |
|
| Submission date |
Dec 28, 2018 |
| Last update date |
Sep 30, 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) |
| GSE124465 |
Lamin A/C promotes DNA base excision repair (human arrays) |
| GSE124467 |
Lamin A/C promotes DNA base excision repair |
|
