|
| Status |
Public on Jun 19, 2014 |
| Title |
Light_ctrl_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
RAW264.7 cells, RNA with monosomes and light polysomes, untreated
|
| Organism |
Mus musculus |
| Characteristics |
cell type: RAW264.7 macrophages
agent: untreated
rna fraction: light
|
| Treatment protocol |
RAW264.7 cells were treated with LPS (100 ng/ml, Sigma, L2630, E. coli serotype O111:B4) for 1 h.
|
| Growth protocol |
RAW264.7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (PAA Laboratories), 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin (all PAN Biotech) at 37°C in 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with Phenol-Chloroform-Isoamylalcohol (PCI, 25:24:1) and precipitated with Isopropanol.
|
| Label |
biotin
|
| Label protocol |
Labeling was performed according to the GeneChip WT Terminal Labeling and Hybridization User Manual, using random hexamers to avoid any bias due to poly-A tail length.
|
| |
|
| Hybridization protocol |
Hybridization was performed according to the GeneChip WT Terminal Labeling and Hybridization User Manual, using a Hybridization Oven 640 and the Fluidiq Station 450 for washing and staining.
|
| Scan protocol |
Scanning was performed with a Genechip scanner 3000 7G.
|
| Description |
L1
After sucrose density gradient centrifugation, ligth RNA was isolated from the fractions containing monosomes, disomes and trisomes. Out of the pooled fractions, 0.7% of the volume was used.
|
| Data processing |
Probe sequences of pm probes were retrieved using the Bioconductor packages oligo (version 1.22.0) and pd.mogene.1.0.st.v1. Probes were mapped to the mouse RefSeq transcriptome as downloaded from the UCSC Genome Browser mm10 refGene table on February 5, 2013. Probes that match transcripts of more than one gene (as defined by a common gene symbol) were excluded. For mapping and further processing of probe information, the Bioconductor packages seqinr and Biostrings and in-house developed Perl scripts were used. Expression values were obtained and summarized at the gene level with the basicRMA() function of the oligo package and the target genes as probe set names. Index, target gene and probe sequence of all pm probes in the analysis are provided in the file mogene10st_annotation.csv. The different fractions (cytoplasmic, free, 40S-associated, light and heavy) were pre-processed as groups. To obtain the proportion of each mRNA (without distinction of isoforms) in a specific pool, expression values (not log2-transformed) were corrected for the relative volume of the pool that was used for labeling. For further analysis, only protein-coding genes with at least four specific probes and log2 expression values above 4 in the cytoplasmic samples of treated and untreated cells were included.
|
| |
|
| Submission date |
Nov 18, 2013 |
| Last update date |
Jun 19, 2014 |
| Contact name |
Johanna Daniela Schott |
| E-mail(s) |
[email protected]
|
| Organization name |
Heidelberg University
|
| Department |
Mannheim Institute for Innate Immunoscience
|
| Lab |
Stoecklin lab
|
| Street address |
Ludolf-Krehl-Str. 13-17
|
| City |
Mannheim |
| ZIP/Postal code |
68167 |
| Country |
Germany |
| |
|
| Platform ID |
GPL17940 |
| Series (2) |
| GSE52449 |
Translational regulation of specific mRNAs controls feedback inhibition and survival during macrophage activation [array] |
| GSE52451 |
Translational regulation of specific mRNAs controls feedback inhibition and survival during macrophage activation |
|
