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Status |
Public on Mar 24, 2013 |
Title |
Q2.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: Quiescence induced by serum depletion
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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 |
Nov 30, 2012 |
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) |
GSE42509 |
Transcriptome-profiling (RNA-seq) and Ribosome-profiling (Ribo-seq) in proliferation, quiescence, senescence and transformed states. |
GSE45833 |
p53 induces transcriptional and translational programs to supress cell proliferation and growth |
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Relations |
SRA |
SRX207948 |
BioSample |
SAMN01821859 |
Supplementary file |
Size |
Download |
File type/resource |
GSM1047587_Q2_Tx_alignment_reads_coverage_per_position_bothStrands_SF_1.65_.wig.gz |
16.3 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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