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Status |
Public on Jul 18, 2011 |
Title |
EGF replicate 3 |
Sample type |
SRA |
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Source name |
Cervical cancer cell line (HeLa)
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Organism |
Homo sapiens |
Characteristics |
treatment: EGF biological replicate: 3 cell line: HeLa cell type: Cervical cancer cell line
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Treatment protocol |
For treatments, the cells were transferred to 60 mm dishes and cultured for 48h in complete growth medium, after which they were starved for 24h in DMEM containing 2% FBS after which they were grown in complete growth medium or the same supplemented with EGF (150 ng/ml) during 6 hours
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Growth protocol |
Cells were cultured at 37ºC in a 95/5 Air/CO2 water saturated atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat inactivated foetal bovine serum (FBS), 2 mM L-glutamine and 100U/ml Penicilin/streptomycine (complete growth medium).
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA from HeLa cells was extracted using the RNeasy RNA Isolation kit (Qiagen) followed by treatment with RNase-free DNase I (Ambion, Austin, TX) following manufacturer's instructions. RNA quantification was performed with a Nanodrop spectrophotometer, and the RNA integrity was assessed using the Bioanalyzer 2100 nanoelectrophoresis Lab-on-a–chip system (Agilent, Wilmington, DE). library selection: 18 nt tag selection by restriction enzyme digestion (DpnII), oligo-dT selection of most 3' fragments, ligation of adapter with MseI site, followed by type II restriction enzyme cut (MseI) library construction protocol: 2 ug of total RNA were used following Illumina's protocol for sequencing of DGE tags. Briefly, libraries of cDNA fragments were generated by capturing transcripts on oligo-dT beads, followed by synthesis of first and second strand cDNA in situ. Cleavage with DpnII resulted in recovery of the most 3' portion of the cDNA molecules, still attached to beads. A 5' adaptor containing a cut site for the type II restriction endonuclease MmeI was ligated to the cDNA. Cleavage with MmeI released fragments of 17-18 bp from the beads. Following 3' adapter ligation, the resulting library was enriched by PCR amplification (15 cycles), and purified by PAGE. planned read length (bp): 36
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina Genome Analyzer |
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Description |
HeLa cells, serum deprived for 24 hours, followed by culture in complete medium supplemented with EGF for 6 additional hours. Biological replicate 3 of 3
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Data processing |
Raw sequence base calls and probability scores (.srf files generated from _seq.txt and _prb.txt files): Raw data were processed using the Illumina pipeline version 0.3.0. removal of adaptor sequences (.fa files): 3’ adapters were recognized and trimmed using a perl script that penalizes mismatches to a lesser extent at read ends, following the distribution of sequencing errors along Solexa reads (Dohm et al. 2008). Where no adaptor could be recognized, sequence was cut down to 18 nt. mapping to RefSeq (_seq.txt files): The compiled collection of expression tags with removed adapters was aligned against the reduced-complexity set of RefSeq entries generated by in silico identification of DpnII cut sites and retrieval of 30 nt sequences on either side of each cut site. The mapping was performed by applying Eland iteratively in order to include all possible product sizes. raw transcript counts (_nonfiltered_tab.txt file): Reads mapping unambiuously were counted for each unique transcript within the reduced-complexity RefSeq reference set. File available as supplementary file on the Series record. per gene transcript counts (_collapsed_tab.txt file): Raw transcript counts were first filtered by removal of RefSeq probes with values smaller than 'mean minus standard error' in at least 90% of the samples, where 'mean = average counts of RefSeq probes corresponding to the same gene within one sample' and 'standard error = standard error of counts of RefSeq probes corresponding to the same gene within one sample' Normalized counts of RefSeq probes corresponding to the same gene (defined by gene symbol) were summed up. Subsequently, counts were normalized by making sample-wise total numbers of reads equal to the median total number of reads for all samples. Finally, normalized counts of RefSeq probes corresponding to the same gene (defined by gene symbol) were summed up. File available as supplementary file on the Series record.
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Submission date |
Aug 18, 2009 |
Last update date |
May 15, 2019 |
Contact name |
Lauro Sumoy |
E-mail(s) |
lsumoy@igtp.cat
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Organization name |
IGTP
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Department |
High Content Genomics and Bioinformatics
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Street address |
Ctra. Can Ruti, Camí de les escoles s/n
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City |
Badalona |
State/province |
Barcelona |
ZIP/Postal code |
08916 |
Country |
Spain |
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Platform ID |
GPL9052 |
Series (1) |
GSE17403 |
Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis |
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Relations |
SRA |
SRX018553 |
BioSample |
SAMN00010724 |
Supplementary file |
Size |
Download |
File type/resource |
GSM442021_s_8_3E6_seq.fa.gz |
112.7 Mb |
(ftp)(http) |
FA |
GSM442021_unambigous_RefSeq_s_8_3E6_seq.txt.gz |
139.5 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
Processed data provided as supplementary file |
Processed data are available on Series record |
Raw data are available in SRA |
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