|
| Status |
Public on Aug 25, 2011 |
| Title |
S2 RNAi-eater no bacteria 30min rep3 |
| Sample type |
RNA |
| |
|
| Source name |
S2 cells, eater-N RNAi, no bacteria, 30 minutes 26.5˚C
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: Schneider line 2 (S2) cells
knockdown: eater-N RNAi
treatment: no bacteria
time: 30 minutes
|
| Treatment protocol |
For RNA interference (RNAi) 10^6 S2 cells were soaked with 15 µg double stranded RNAs as described (Chung & Kocks 2011 J Biol Chem 286:26524-26532). After 60 to 72 hours, cells were shifted to 0˚C (temperature non-permissive for phagocytosis). FITC-labeled, heat-inactivated Gram-positive and Gram-negative bacteria (Staphylococcus aureus and Serratia marcescens, respectively) were added; bacterial input was previously optimized to achieve 50% of cells binding to both kind of bacteria. Bacteria were briefly centrifuged onto the cells and incubated for 30 minutes on ice to maximize binding to cells. Cultures were shifted to 26.5˚C (temperature permissive for phagocytosis) and RNA was extracted at three different timepoints: 30 or 90 or 180 minutes at 26.5˚C. Parallel samples were analysed in all cases in phagocytosis assays with S. aureus and S. marcescens to confirm the expected biological effect of eater knockdown (betweeen 50 to 80% decreased phagocytosis).
|
| Growth protocol |
Drosophila Schneider line 2 (S2) cells were maintained in the exponential growth phase at 26.5˚C in Schneider's Drosophila Medium (Invitrogen) containing 10% fetal bovine serum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA (10 to 20 µg per sample) was extracted according to the manufacturer's instructions using the RNeasy Plus Kit (Qiagen). RNA integrity was confirmed with a Agilent Bioanalyzer (Nano Kit).
|
| Label |
biotin
|
| Label protocol |
Samples were processed by the Biopolymers Core Facility at Harvard Medical School using Full Affymetrix Service according to standard Affymetrix protocols. Complete sets of biological replicates (A-series, B-series, C-series) were processed in parallel to minimize technical day-to-day variations. cRNA: biotin-labled via in vitro transcription
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| |
|
| Hybridization protocol |
Affymetrix standard protocol. Hybridization: 16 hour incubation; Wash & Stain: double amplified with phycoerythrin-tagged streptavidin
|
| Scan protocol |
Affymetrix standard protocol. Scan GeneChip: exitation: 488 nm, emission: 570 nm
|
| Description |
gene expression data from eater RNAi-treated S2 cells in the absence of bacteria
|
| Data processing |
Data were analysed with freely available GenePattern version 2.0 Software on the BROAD public Server using CEL files as input. Background subtraction and normalization were performed with ExpressionFileCreator using GC-RMA and quantile normalization. Data were not filtered (no threshold/ceiling filters, no variance filters). Normalized data were preprocessed using MultiplotPreprocess (no replicate FC, no random data, no outlier elimination, but with Stats), analysed using Multiplot and means calculated from 3 independent biological replicates were visualized with scatter plots. Probability of differential expression was calculated by Multiplot from 3 independent biological replicates per sample using FDR (Hochberg) p-values adjusted for multiple hypothesis testing; probability of ≤ 0.05 was considered significant. [For quality control purposes, data were analysed with Microarray Suite version 5.0 (MAS 5.0).]
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| |
|
| Submission date |
Aug 22, 2011 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Christine Kocks |
| E-mail(s) |
[email protected]
|
| Organization name |
Massachusetts General Hospital
|
| Department |
Pediatrics/Molecular Biology
|
| Lab |
Kocks
|
| Street address |
185 Cambridge St CPZN7.250
|
| City |
Boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02446 |
| Country |
USA |
| |
|
| Platform ID |
GPL1322 |
| Series (1) |
| GSE31564 |
Gene expression response to bacterial phagocytosis by S2 cells (control and eater RNAi knock down) |
|
| Relations |
| Reanalyzed by |
GSE119084 |
