|
| Status |
Public on May 01, 2012 |
| Title |
Adaptive Sample (0.1Gy + 2Gy) at 4h; biological repl. B |
| Sample type |
RNA |
| |
|
| Source name |
Adaptive Sample (0.1Gy + 2Gy) at 4h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: WI-38
cell type: Primary fetal human lung fibroplasts (TP53 proficient)
age: 12 FW
|
| Biomaterial provider |
http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=AG06814
|
| Treatment protocol |
Cells were exposed to a challenging dose of 2 Gy of X-rays (160 kVp) 4 hours after receiving a sham exposure (0 Gy) or an adaptive dose of 0.1 Gy.
|
| Growth protocol |
Primary fetal human lung fibroplasts WI-38 (Coriell; AG06814) were cultured under physiological oxygen condition (3% O2 and 10% CO2) in DMEM medium with 10 % serum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Extraction of total RNA was performed according to the manufacturer's instructions. An initial step to remove ribosomal RNA was used to minimize background and to increase detection sensitivity and specificity. Ribosomal RNA subtraction was conducted using a protocol that was modified by Affymetrix for the RiboMinus Transcriptome Kit (Invitrogen).
|
| Label |
biotin
|
| Label protocol |
Double-stranded cDNA was synthesized with random hexamers tagged with a T7 promoter sequence and used as a template in the presence of T7 RNA polymerase to produce cRNA. In the second cycle of cDNA synthesis, random hexamers were used to reverse transcribe cRNA from the first cycle, producing single-stranded DNA (ssDNA) in the sense orientation. The ssDNA was fragmented by the uracil DNA glycosylase and apurinic/apyrimidinic endonuclease 1 which recognizes the dUTP incorporated in the ssDNA during the second-cycle, first-strand reverse transcription reaction and breaks the DNA strand. The fragmented ssDNA was labeled with terminal deoxynucleotidyl transferase and a DNA labeling reagent that is covalently linked to biotin.
|
| |
|
| Hybridization protocol |
The fragmented, biotinylated ssDNA probes (5.5ug) were hybridized in a volume of 220ul at 45°C for 16 hours to Affymetrix high density Human Junction Arrays. The arrays were washed and stained with streptavidinphycoerythrin (SAPE, final concentration 10 μg/ml). Signal amplification was performed using a biotinylated anti-streptavidin antibody.
|
| Scan protocol |
The arrays were scanned on an Affymetrix GeneChip® Scanner 3000 7G scanner with an autoloader, according to the Affymetrix GeneChip® WT Sense Target Labeling Assay protocol for the GeneChip Human Gene 1.0 ST Array.
|
| Description |
total RNA (ribosomal RNA removed)
0.1_2Gy_4h_B_(HuGene-1_0-st-v1).CEL
|
| Data processing |
Robust multi-array analysis (RMA) was performed to normalize data collected from the different samples through R bioconductor (v.2.13.0). Samples were examined for quality control using the NUSE protocol.
|
| |
|
| Submission date |
Apr 30, 2012 |
| Last update date |
May 01, 2012 |
| Contact name |
Bahram Parvin |
| E-mail(s) |
B_Parvin@lbl.gov
|
| Organization name |
Lawrence Berkeley National Laboratory
|
| Lab |
Parvin
|
| Street address |
M.S. 977
|
| City |
Berkeley |
| State/province |
Ca |
| ZIP/Postal code |
94720 |
| Country |
USA |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE37668 |
Time-varying expression data from WI38 cell lines that were exposed to a challenge dose with or without a priming dose |
|
