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Sample GSM538820 Query DataSets for GSM538820
Status Public on Apr 29, 2010
Title HEK 293 cells, top 25 miRNAs inhibited, siRNA-transfected, replicate 1
Sample type RNA
 
Source name HEK 293, miRNA siRNAs
Organism Homo sapiens
Characteristics cell line: HEK 293
sirna: 25 miRNAs
Treatment protocol siRNA, miRNA and 2'-O-methyl oligonucleotide transfections of HEK 293 T-REx Flp-In cells were performed in 6-well format using Lipofectamine RNAiMAX (Invitrogen) as described by the manufacturer. Detailed experimental procedures can be found in the Supplement to PMID 20371350.
Growth protocol HEK 293 T-REx Flp-In cells (Invitrogen) were grown in D-MEM high glucose with 10% (v/v) fetal bovine serum, 1% (v/v) 2 mM L-glutamine, 1% (v/v) 10,000 U/ml penicillin/10,000 µg/ml streptomycin, 100 µg/ml zeocin and 15 µg/ml blasticidin. Cell lines stably expressing FLAG/HA-tagged proteins were generated by co-transfection of pFRT/TO/FLAG/HA or pFRT/FLAG/HA constructs with pOG44 (Invitrogen). Cells were selected by exchanging zeocin with 100 µg/ml hygromycin. Expression of FLAG/HA-IGF2BP1, -2, -3 and TNRC6A, B and C was induced by addition of 250 ng/ml doxycycline 15 to 20 h before crosslinking. Detailed experimental procedures can be found in the Supplement to PMID 20371350.
Extracted molecule total RNA
Extraction protocol Trizol extraction of total RNA was performed according to the manufacturer's instructions.
Label biotin
Label protocol 2 µg of purified total RNA was used in the One-Cycle Eukaryotic Target Labeling Assay (Affymetrix) according to manufacturer's protocol. Biotinylated cRNA targets were cleaned up, fragmented, and hybridized to Human Genome U133 Plus 2.0 Arrays (Affymetrix).
 
Hybridization protocol 3 µg cRNA was hybridized to HGU133 Plus 2.0 Arrays for 16 h at 45ºC.
Scan protocol GeneChips were scanned on an Affymetrix GeneChip 3000 7G scanner.
Description Gene expression data from HEK 293 cells transfected with a cocktail of 2'OMe oligoribonucleotides inhibiting the top 25 miRNAs.
miRNA inhibition repl 1
Data processing We imported the CEL files into the R software (http://www.R-project.org) using the BioConductor affy package (Gentleman et al., 2004). The transcript probe set intensities were background-corrected, adjusted for non-specific binding and quantile normalized with the GCRMA algorithm (Wu, 2006). Probe sets with more than 6 of the 11 probes mapping ambiguously to the genome were discarded, as were probe sets that mapped to multiple genes. We then collected all probe sets matching a given gene, and we selected for further analysis the RefSeq transcript with median 3'UTR length corresponding to that gene. In total, 16,063 transcripts were identified. The log-intensity of probe sets mapping to the gene were then averaged to obtain the expression level per RefSeq transcript. The changes of transcript abundances were computed as the logarithm of the ratio of transcript expression in the cocktails of siRNA-treated samples and mock-transfected cells. Detailed analysis procedures can be found in the Supplement to PMID 20371350.
 
Submission date Apr 28, 2010
Last update date Aug 28, 2018
Contact name Markus Hafner
E-mail(s) mhafner@rockefeller.edu
Phone +1 212 327 7696
Fax +1212 327 7652
Organization name The Rockefeller University
Department Laboratory of RNA Molecular Biology
Lab Tuschl lab
Street address 1230 York Ave, Box 186
City New York
State/province NY
ZIP/Postal code 10065
Country USA
 
Platform ID GPL570
Series (2)
GSE21577 Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP: miRNA inhibition data
GSE21578 Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP
Relations
Reanalyzed by GSE119087

Data table header descriptions
ID_REF
VALUE log2 GC-RMA signal

Data table
ID_REF VALUE
1007_s_at 7.024979392
1053_at 10.09835382
117_at 2.322077413
121_at 2.747656324
1255_g_at 2.186926124
1294_at 2.186926124
1316_at 2.80556458
1320_at 2.187929871
1405_i_at 2.186926124
1431_at 2.46041675
1438_at 2.195340115
1487_at 7.319598638
1494_f_at 2.188120604
1552256_a_at 10.55556264
1552257_a_at 10.64769993
1552258_at 2.187502697
1552261_at 2.267212354
1552263_at 6.514994536
1552264_a_at 7.949652438
1552266_at 2.188348822

Total number of rows: 54675

Table truncated, full table size 1213 Kbytes.




Supplementary file Size Download File type/resource
GSM538820.CEL.gz 4.8 Mb (ftp)(http) CEL
Raw data provided as supplementary file
Processed data included within Sample table

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