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| Status |
Public on Oct 01, 2013 |
| Title |
Next-Generation Sequencing for Whole Transcriptome Analysis in Scrambled Control and EGFR Knockdown Cells Cultured under Normoxia and Hypoxia |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Purpose: The goal of this study is to identify the mRNA clusters that are regulated by EGFR under normoxia or hypoxia.
Method: Total RNAs were extracted from HeLa cells expressing scrambled control or EGFR shRNA-E1 that cultured under normoxia or hypoxia (1% O2) for 24h. Customized Next-Generation RNA Deep Sequencing, including both small RNA application and whole transcriptome analysis, was performed according to the standard procedure instructed by Applied Biosystems. For whole transcriptome analysis, SOLiD fragment colorspace transcriptome reads (50nt) were mapped to the human genome (hg19) and assigned to ensemble transcripts using Bioscope 1.3.1 (Life Technologies). The values of reads per kilobase per million reads (RPKM) were determined by Bioscope 1.3.1 CountTags tool using default parameters. Primary alignments with a minimum mapping quality of 10 and minimum alignment score of 10 were counted.
Results: Deep sequencing analysis identified subclasses of mRNAs that were affected by EGFR either under normoxia or hypoxia. EGFR-regulated mRNAs (with Log2 fold-change affected by EGFR ≥ 0.4 or ≤ -0.4) were sorted and over-lapped with mRNAs that were targeted (based on published data and TargetScan prediction with total context score ≤ -0.20) by the top miRNA candidates affected by EGFR under hypoxia, resulting in 439 mRNAs that regulated by EGFR and likely targeted by the miRNA candidates in response to hypoxia.
Conclusion: Whole transcriptome analysis revealed a novel cluster of mRNAs that are likely regulated by EGFR through miRNAs in response to hypoxic stress.
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| Overall design |
RNA profiles of HeLa cells expressing scrambled control (S) or EGFR shRNA-E1 (A1) that cultured under normoxia or hypoxia (1% O2) for 24h were generated by AB SOLiD curstomarized next-generation sequencing, including both small RNA application and whole transcriptome analysis. S: HeLa expressing scrambled control cultured under normoxia; A1: HeLa expressing EGFR shRNA-E1 cultured under normoxia; HS: HeLa expressing scrambled control cultured under hypoxia for 24h; HA1: HeLa expressing EGFR shRNA-E1 cultured under hypoxia for 24h. In total, 4 biological samples with no replicates resulted in 4 whole transcriptome RNA profiles.
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| Contributor(s) |
Shen J, James BP, Liu X, Liu C, Hung M |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
| Submission date |
Mar 01, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Mien-Chie Hung |
| E-mail(s) |
mhung@mdanderson.org
|
| Organization name |
MD Anderson Cancer Center
|
| Street address |
1515 Holcombe Boulevard
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platforms (1) |
| GPL13393 |
AB SOLiD 4 System (Homo sapiens) |
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| Samples (4)
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| This SubSeries is part of SuperSeries: |
| GSE44804 |
Scrambled Control and EGFR Knockdown Cells Cultured under Normoxia and Hypoxia |
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| Relations |
| BioProject |
PRJNA191817 |
| SRA |
SRP019229 |
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