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| Status |
Public on Nov 27, 2024 |
| Title |
A2 |
| Sample type |
RNA |
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| Source name |
SAEC cells exposed to CSC
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| Organism |
Homo sapiens |
| Characteristics |
genotype: Wild-type
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| Treatment protocol |
Cigarette smoke and drug exposures: After being prepared as previously mentioned , cigarette smoke condensates (CSC) generated from Kentucky Reference 1R4F research blend cigarettes (University of Kentucky) were resuspended in DMSO at a stock concentration of 25 mg tar/ml. Cells were grown in 10-cm plates in suitable normal media (NM) containing DMSO or NM containing CSC (0.025mg/ml) for smoke exposure experiments. Daily medium changes included the inclusion of brand-new CSC or DMSO control. Cells were collected for analysis at different times after being subcultured as needed. The DNA demethylating agent, decitabine (MilliporeSigma, St. Louis, MO) was added in the culture medium daily (100nM x 72hrs). Mithramycin was obtained from Sigma. Methysticin (MCE) was obtained from MilliporeSigma, St. Louis, MO. Cells were cultivated in NM containing or lacking mithramycin or methysticin for drug exposure treatments. After changing the media and adding mithramycin or methysticin for 24 hours at the recommended concentrations, the cells were harvested for additional analysis at the designated times. NFkB-P65 siRNA (#6261S /Cell Signaling) was used to deplete NFkB in relative experiments.
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| Growth protocol |
All lung cancer cell lines from The American Type Culture Collection (ATCC; Manassas, VA) were kept in RPMI media supplemented with 10% FBS, 10 mM of glutamic acid, and 1% penicillin/streptomycin (normal media). Primary normal human small airway epithelial cells (SAEC) from Lonza, Inc. (Frederick, MD) were cultured following the vendor’s instructions. The immortalized human bronchial epithelial cells (HBEC) generously offered by John D. Minna (U-T Southwestern, Dallas, TX) were cultivated according to the instructions (30). All cells were routinely checked for mycoplasma using a Sigma kit (Cat. no. MP0025) and confirmed by HLA typing to match their original stocks.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from each sample was quantified using the Nanodrop 1000 and the RNA integrity was assessed by Agilent 2100 Bioanalyzer. About 5 μg total RNA of each sample was used for labeling and array hybridization as the following steps: 1) Reverse transcription with invitrogen Superscript ds-cDNA synthesis kit; 2) ds-cDNA labeling with Nimblegen one-color DNA labeling kit; 3) Array hybridization using the NimbleGen Hybridization System and followed by washing with the Nimblegen wash buffer kit. 4) Array scanning using the Axon GenePix 4000B microarray scanner (Molecular Devices Corporation).
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| Label |
Cy3
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| Label protocol |
Labeling was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
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| Hybridization protocol |
Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA following their standard operating protocol. See www.nimblegen.com.
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| Scan protocol |
Scanning was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
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| Data processing |
Scanned images (TIFF format) were then imported into NimbleScan software (version 2.5) for grid alignment and expression data analysis. Expression data were normalized through quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software. The Probe level (*_norm_RMA.pair) files and mRNA level (*_RMA.calls) files were generated after normalization. The 4 mRNA level files were imported into Agilent GeneSpring Software (version 11.0) for further analysis. lncRNAs and mRNAs that at least 2 out of 4 samples have values greater than or equal to lower cut-off: 50.0 (“All Targets Value”) were chosen for data analysis. Differentially expressed lncRNAs and mRNAs were identified through Fold Change filtering. Pathway Analysis and GO analysis were applied to determine the roles of these differentially expressed mRNAs played in these biological pathways or GO terms. Finally, Hierarchical Clustering was performed to show distinguishable lncRNA and mRNA expression profiling among samples.
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| Submission date |
Nov 26, 2024 |
| Last update date |
Nov 27, 2024 |
| Contact name |
Sichuan Xi |
| Organization name |
NCI/NIH
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| Street address |
Building 10
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL35135 |
| Series (1) |
| GSE282877 |
lncRNA/mRNA expression profiling for 4 Human RNA samples |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM8651951_A2_532.pair.gz |
1.9 Mb |
(ftp)(http) |
PAIR |
| GSM8651951_A2_532_RMA.calls.gz |
498.2 Kb |
(ftp)(http) |
CALLS |
| GSM8651951_A2_532_norm_RMA.pair.gz |
2.0 Mb |
(ftp)(http) |
PAIR |
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