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| Status |
Public on Feb 06, 2014 |
| Title |
Wild Type Staufen associated, RNase treated mRNA |
| Sample type |
SRA |
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| Source name |
HEK293T cells transfected with pChStaufen-TAP
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
transfected with: pChStaufen-TAP
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| Treatment protocol |
HEK293T cells were transfected with DNA plasmids using the calcium phosphate method (Wigler et al, 1979). Plasmids pChStaufen-TAP, pChMut-TAP and pC-TAP as well as rabbit and rat antibodies specific for hStau1 have been previously described (de Lucas et al, 2010; Villacé et al, 2004).
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| Growth protocol |
The HEK293T (DuBridge et al, 1987) cell line was cultured as previously described (Villacé et al, 2004)
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| Extracted molecule |
total RNA |
| Extraction protocol |
TAP-associated RNAs were obtained by treatment of Calmodulin-agarose resin with 0.2 mg/ml Proteinase K and 0.5% SDS in TNE for 1 hour at 37C. After phenol extraction, RNAs were precipitated with 2 ethanol volumes and 20 μg of glycogen (Roche) and resuspended in DEPC-treated H2O. Before cDNA synthesis, each RNA preparation was monitored using the Agilent 2100 Bioanalyzer (Agilent technologies).
RNA samples of hStau1-TAP (treated or not with RNase T1) and the untreated hStau1-Mut (100 ng each) were used to generate cDNA libraries according to Illumina mRNA-seq protocol at Fasteris SA Company (Plan-Les-Ouates, Switzerland) but the poly (A+) RNA purification step was skipped. Furthermore, mRNA fragmentation was omitted in the T1-RNase treated hStau1-associated RNA, as it already contained RNAs about 100 nt in size. cDNA libraries were subjected to ends repair reaction and Illumina adapter ligation and further purified from polyacrilamide gels. This purification step excluded the identification of putative small RNAs present in the samples. cDNA libraries were sequenced at Fasteris SA in an Illumina HiSeq2000 sequencer. Reads length were 100nt in a single-read run cycle.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
hStau1_WT_RNase
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| Data processing |
Filtering raw reads: Removal of illumina adaptors; removal of rRNA sequences by BWA aligning against representative human ribosomal RNAs (28S, 18S, 5.8S, 5S)
Filtered reads alignment: Clean reads were aligned to human genome (hg19) with BWA with default parameters
Counts per gene: After alignment, the number of reads that matched each known gene was calculated using countOverlaps function of GenomicFeatures package (www.bioconductor.org). Gene annotations and coordinates were obtained from UCSC database using the “RefGene” table.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include read counts per gene and per sample.
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| Submission date |
May 23, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Juan Carlos Oliveros |
| Organization name |
CNB, CSIC
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| Street address |
Darwin 3
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| City |
Cantoblanco |
| State/province |
Madrid |
| ZIP/Postal code |
28049 |
| Country |
Spain |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE47226 |
Functional signature for the recognition of specific target mRNAs by human staufen1 protein |
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| Relations |
| SRA |
SRX286279 |
| BioSample |
SAMN02178354 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1147076_hStau1_WT_RNase_counts.txt.gz |
1.2 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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