shRNA sequence: none cell line: HeLa treatment: control
Treatment protocol
Knockdowns of RBM5, RBM6 or RBM10 in Hela cells were performed by lentiviral infections using the pLKO.1 carrier vector for specific shRNA (Sigma Aldrich).
Growth protocol
HeLa cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin.
Extracted molecule
total RNA
Extraction protocol
RNA from three biological replicates of sh-RBM infected HeLa cells was purified using Rneasy mini kit (Qiagen).
Label
biotin
Label protocol
Samples were enzymatically fragmented and biotinylated using the WT Terminal Labeling Kit (Affymetrix)
Hybridization protocol
Samples were hybridized using Affymetrix hybridization kit materials. Heat cocktails at 99° for 5 minutes, then 42° for 5 minutes centrifuge at max speed for 1 minute (N.B. this deviates from Affy SOP). Transfer 200μl of hyb solution to each array, then tape holes and parafilm. Hybridize 16 hours at 45° at 60rpm. Fluidics washing is FS450_0001.
Scan protocol
Affymetrix Gene ChIP Scanner 3000 7G
Description
none
Data processing
Affymetrix HJAY dataset analysis was performed by GenoSplice technology (www.genosplice.com). Raw data was processed using Expression Console to get the RMA intensities values of Affymetrix HJAY probesets. Additional analysis included the follwing. Data were normalized using quantile normalization. Background corrections were made with antigenomic probes and probes were selected according to their %GC, cross-hybridization status and potential overlap with repeat region as previously described [PMID:23321315, PMID:23284676]. Only probes targeting exons and exon-exon junctions annotated from FAST DB® transcripts (release fastdb_2012_2) were selected [PMID:16052034,PMID:17547750]. Only probes with a DABG Pvalue ≤ 0.05 in at least half of the arrays were considered for statistical analysis [PMID:23321315, PMID:23284676]. Only genes expressed in at least one compared condition were analyzed. To be considered to be expressed, the DABG Pvalue had to be ≤ 0.05 for at least half of the gene probes. We performed an unpaired Student’s t-test to compare gene intensities between two experimental conditions. Genes were considered significantly regulated when fold-change was ≥1.5 and Pvalue 0.05 (unadjusted P-value). Analysis at the splicing level was first performed taking into account only exon probes (EXON analysis) in order to potentially detect new alternative events that could be differentially regulated (i.e., without taking into account exon-exon junction probes). Analysis at the splicing level was also performed by taking into account exon-exon junction probes (SPLICING PATTERN analysis) using the FAST DB® splicing patterns annotation (i.e., for each gene, all possible splicing patterns were defined and analyzed. All types of alternative events can be analyzed: Alternative first exons, alternative terminal exons, cassette exon, mutually exclusive exons, alternative 5’ donor splice site, alternative 3’ acceptor splice sites and intron retention). EXON and SPLICING PATTERN analyses were performed using unpaired Student's t-test on the splicing-index as previously described [PMID:23321315, PMID:23284676]. Results were considered statistically significant for unadjusted P-values ≤ 0.05 and fold-changes ≥ 2.0 for EXON analysis and for unadjusted P-values ≤ 0.05 and fold-changes ≥ 1.5 for SPLICING PATTERN analysis. Gene Ontology (GO), KEGG and REACTOME analyses of differentially regulated genes were performed using DAVID [PMID:19131956].
Submission date
May 28, 2013
Last update date
Dec 21, 2013
Contact name
Endre Sebestyén
Organization name
Semmelweis University
Department
1st Department of Pathology and Experimental Cancer Research
