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| Status |
Public on Jun 01, 2013 |
| Title |
C.rp |
| Sample type |
SRA |
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| Source name |
Immortalized primary fibroblasts
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| Organism |
Homo sapiens |
| Characteristics |
cell line: BJ cells
condition: Control sample
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| Growth protocol |
Immortalized human BJ primary fibroblast cells (by hTERT expression) were cultured in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS) in 5% CO2 at 37 °C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were treated with cycloheximide (100 μg/ml) for 8-10 minutes, washed with ice-cold PBS (cycloheximide, 100 μg/ml), pelleted, and lysed in buffer A (20 mM Tris-HCl, pH7.8, 100mM KCl, 10mM MgCl2, 1% Triton X-100, 2mM DTT, 100 μg/ml cycloheximide, 1x complete protease inhibitor). Lysates were centrifuged at 5000 rpm and the supernatant was treated with 2U/μl of RNase I (Ambion) for 40 minutes at room temperature. Lysates were fractionated on a linear sucrose gradient (7%-47%) using the SW-41Ti rotor at 36,000 rpm for 2hrs. Fractions enriched in monosomes were polled and treated wih proteinase K (Roche) in 1%SDS. Released RNA fragments were purified using trizol and precipitated in the presence of glycogen.
RNA from the pooled monosomes was gel-purified on a denaturing 10% polyacrylamide urea (7M) gel. A section corresponding to 30-33 nucleotides, the region where most of the ribosome-protected fragments are comprised, was excised, eluted, and ethanol precipitated. The resulting fragments were 3′-dephosphorylated using T4 PNK (NEB) for 6 h at 37 °C in MES buffer (100 mM MES-NaOH, pH 5.5, 10 mM MgCl2, 10 mM β-mercaptoethanol, 300 mM NaCl). 3′ adaptor was added with T4 RNA ligase 1 (NEB) for 2.5h at 37°C. Ligation products were 5′-phosphorylated with T4 PNK for 30 min at 37°C. 5′ adaptor was added with T4 RNA ligase 1 for 18h at 22°C.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
ribosome-protected fragments
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| Data processing |
Reads were aligned to a reference set of human transcripts using Bowtie
Number of reads mapping to each transcript was counted and normalized to FPKMs
Supplementary_files_format_and_content: wig files represent read coverage at each genomic location (hg19), normalized to 10M reads
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| Submission date |
Apr 04, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Ran Elkon |
| Organization name |
Tel Aviv University
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| Department |
Human Genetics
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| Street address |
Ramat Aviv
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| City |
Tel Aviv |
| ZIP/Postal code |
69494 |
| Country |
Israel |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE45785 |
Transcriptome-profiling (RNA-seq) and Ribosome-profiling (Ribo-seq) of BJ cells treated with Nutlin-3a, an MDM2 inhibitor, which induces p53. |
| GSE45833 |
p53 induces transcriptional and translational programs to supress cell proliferation and growth |
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| Relations |
| SRA |
SRX260245 |
| BioSample |
SAMN01999142 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1115204_Nut.0h_Tx_alignment_reads_coverage_per_position_bothStrands_SF_1.11_.wig.gz |
36.0 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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