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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 28, 2012 |
| Title |
mRNA-seq for rapamycin treated PC3 cells, replicate #1. |
| Sample type |
SRA |
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| Source name |
PC3 human prostate cancer cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: PC3
sample type: polyA RNA
treatment: rapamycin
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
PC3 cells were treated with rapamycin (50 nM - Calbiochem) or PP242 (2.5 µM - Intellikine) for 3 hours. Cells were subsequently treated with cycloheximide (100 µg/mL - Sigma) and detergent lysis was performed in the dish. The lysate was treated with DNase and clarified, and a sample was taken for RNA-Seq analysis. Lysates were subjected to ribosome footprinting by nuclease treatment. Ribosome-protected fragments were purified, and deep sequencing libraries were generated from these fragments, as well as from poly(A) mRNA purified from non-nuclease treated lysates. These libraries were analyzed by sequencing on the Illumina GAII.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
3hr rapamycin treated human PC3 prostate cancer cells.
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| Data processing |
Each sequencing run resulted in approximately 20-25 million raw reads per sample, of which 5-12 million unique reads were used for subsequent analysis. Ribosome footprint and RNA-Seq sequencing reads were aligned against a library of transcripts from the UCSC Known Genes database GRCh37/hg19. The first 25 nucleotides of each read were aligned using Bowtie and this initial alignment was then extended to encompass the full fragment-derived portion of the sequencing read while excluding the linker sequence. Read density profiles were then constructed for the canonical transcript of each gene, using only reads with 0 or 1 total mismatches between the read sequence and the reference sequence, comprised of the transcript fragment followed by the linker sequence. Footprint reads were assigned to an A site nucleotide at position +15 to +17 of the alignment, based on the total fragment length; mRNA reads were assigned to the first nucleotide of the alignment. The average read density per codon was then computed for the coding sequence of each transcript, excluding the first 15 and last 5 codons, which can display atypical ribosome accumulation.
Average read density was used as a measure of mRNA abundance (RNA-Seq reads) and of protein synthesis (ribosome profiling reads). For most analyses, genes were filtered to require at least 256 reads in the relevant RNA-Seq samples. Translational efficiency was computed as the ratio of ribosome footprint read density to RNA-Seq read density, scaled to normalize the translational efficiency of the median gene to 1.0 after excluding regulated genes (log2 fold-change +/- 1.5 after normalizing for the all-gene median). Changes in protein synthesis, mRNA abundance, and translational efficiency were similarly computed as the ratio of read densities between different samples, normalized to give the median gene a ratio of 1.0. This normalization corrects for differences in the absolute number of sequencing reads obtained for different libraries. 5333 (replicate #1) and 3977 (replicate #2) unique mRNAs passed a preset read threshold of 256 reads for single-gene quantification for all treatment conditions.
processed data file contains per gene read count, columns: 1) UCSC ID, 2) Length of trimmed CDS, 3) Read count in trimmed CDS
Genome Build:
mrna_rapa_a_qexpr.txt: UCSC GRCh37/hg19
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| Submission date |
Feb 01, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Andrew Caleb Hsieh |
| E-mail(s) |
andrew.hsieh@ucsf.edu
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| Phone |
415-514-4827
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| Fax |
415-514-4826
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| Organization name |
UCSF
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| Department |
Urology
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| Lab |
Davide Ruggero
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| Street address |
1450 3rd Street, HD Rm 319
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (1) |
| GSE35469 |
The translational landscape of mTOR signaling steers cancer initiation and metastasis |
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| Relations |
| SRA |
SRX118287 |
| BioSample |
SAMN00779919 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM869038_mrna_rapa_a_qexpr.txt.gz |
132.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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