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| Status |
Public on Apr 09, 2015 |
| Title |
KPC1 rep2- run1 |
| Sample type |
SRA |
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| Source name |
KPC1 - run1
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| Organism |
Homo sapiens |
| Characteristics |
cell type: U87-MG glioblastoma human cells
transfected with: vectors coding for Myc-KPC1
xenografts host mice: 7-wks old male Balb/C nude mice
tumor stage: 21 days old xenografts
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| Treatment protocol |
Exponentially growing U87-MG cells were stably transfected with an empty vector (V0) or vectors coding for Myc-KPC1 or Flag-p50. Cells were dissociated with trypsin, washed with PBS, and brought to a concentration of 50×106 cells/ml. Cell suspension (5×106/0.1 ml) was inoculated subcutaneously at the right flank of 7-weeks old male Balb/C nude mice (n=7).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Following 21 days, mRNA from U87-MG xenografts was isolated using an RNA purification kit (NucleoSpin® Kit for RNA purification, was from Macherey-Nagel)
Total mRNA was analyzed using Illumina HiSeq 2500. Standard Illumina protocols were used to prepare the RNA libraries.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Sample 5
processed data file: genes.fpkm_tracking, final_results_U87_run1+run2.xlsx
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| Data processing |
both analysis: RNA was analyzed using Illumina HiSeq 2500. The number of reads was between 25,949,993 and 39,809,768 per sample
RNASeq analysis mapped to the human genome:
The reads were mapped to the human genome (GRCh37) using Tophat version 2.0.9 , Up to 3 mismatches were allowed per read, with up to 3 mismatches per segment.The-b2-sensitive parameter was set.
Only uniquely mapped reads were counted in the analysis, using the HTSeq-count package version 0.5.3p3 with ‘intersection-nonempty’ mode
The counts normalization and the differential expression analysis were done using the DESeq2 package version 1.2.8
The reads were mapped both to the human and mouse genomes (builds GRCh37.64 and NCBIM37.64, respectively) using Bowtie2 version 2.2.2 with the parameters-sensitive - k 5.
Using an in house written Perl script, we determined for each read whether it was mapped to both, one, or neither of the genomes. We used only reads that were uniquely mapped to the mouse genome.
Gene expression values were determined using the Cufflinks software version 2.2.1 by the "cuffnorm" command.
Genome_build: GRCh37 for the human trascripts analysis. GRCh37.64 (human) and NCBIM37.64 (murine) builds were used in the murine trascripts analysis
Supplementary_files_format_and_content: final_results_U87_run1+run2.xlsx: RNASeq analysis mapped to the human genome. A differential expression table for U87-MG samples that contains the following columns: Gene name and chromosomal position for each gene , Base mean – mean of the normalized counts across all samples. For each pair of samples: Log2Fold change – log2 of the fold change between the pair of samples. For comparisons where samples of one conditions are zeroes and the other conditions’ are low (average < 1), the fold change that is calculated by DESeq2 is very high due to division by zero. In such cases the fold change is set to “NA_zeros’. p-value - The p value for rejecting the null hypothesis ’mean ==meanB’. padj - The Benjamini & Hochberg adjusted p values (multiple testing correction). Flag – can be either of the following: Tested – the statistical test was conducted, the log2 fold change and the adjusted p-value are informative. Low count – indicates that either the gene was filtered due to low expression (in such case the adjusted p-value will be 'NA'), or none of the samples in that comparison received 5 counts or more. Outlier:X – this gene was not tested for differential expression in this comparison since it is an outlier in sample X. The p-value and adjusted p-value are 'NA' in this case. All zero – the counts of this gene is 0 in all samples. Such genes are not tested for differential expression. Significant - can be either "yes" or "no", it is “yes” if the adjusted p-value (padj) is < 0.05
Supplementary_files_format_and_content: genes.fpkm_tracking: RNASeq analysis mapped to the murine genome to identify the host immune cell signeture. Contains gene expression values. To identify the cell type origin of the murine reads, gene expression of each sample was compared with the characteristic gene expression of murine immune cell types obtained from the Immunological Genome Project (ImmGen)
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| Submission date |
Aug 19, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Aaron Ciechanover |
| E-mail(s) |
aaroncie@tx.technion.ac.il
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| Phone |
+972-48295427
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| Organization name |
Technion-Israel Institute of Technology
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| Department |
Faculty of Medicine, Cancer and Vascular Biology Research Center
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| Street address |
Efron Street, Bat Galim, POB 9649
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| City |
Haifa |
| State/province |
Haifa |
| ZIP/Postal code |
31096 |
| Country |
Israel |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE60530 |
Profile of gene expression in U87-MG xenografts expressing control vector (V0), the ubiquitin ligase KPC1 or the p50 subunit of the NF-kB transcription factor, using RNASeq analysis of transcripts mapped independently to the human and murine genomes |
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| Relations |
| BioSample |
SAMN02998087 |
| SRA |
SRX683527 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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