|
| Status |
Public on Apr 25, 2013 |
| Title |
Ph RNAi-treated BG3 cells, biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Ph RNAi-treated BG3 cells, 5 days RNAi treatment
|
| Organism |
Drosophila melanogaster |
| Characteristics |
gender: male
|
| Treatment protocol |
For RNAi treatment in BG3 cells, media was replaced with 1 ml of Express Five SFM (Invitrogen) with 1% FCS, (and 10 μg per ml insulin for BG3 cells), for 2 hours. 10 μg of dsRNA was added per well of a 6-well plate. Media was adjusted to 3 ml and 10% FCS withSchneider's media after 2 hrs of RNAi treatment. Cells were replated as needed. Templates for dsRNA synthesis were made by PCR from cDNA or genomic DNA templates using primers with T7 promoters. In most experiments, equal amounts of two dsRNAs against the target were used. Both individual dsRNAs knocked down the targets, but knockdown was generally more efficient with a mixture. Templates were designed to avoid off-target matches of ≥19 nucleotides using Drosophila RNAi Screening Center (http://www.flyrnai.org/) online tools. Ph RNAi constructs target regions of homology between ph-p and ph-d.
|
| Growth protocol |
BG3 cells were cultured in Schneider's media with 10% FCS and 10 μg per ml insulin. Drosophila larvae are grown under standard conditions at 25 degrees.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from wing imaginal discs was isolated using Zymo ZR RNA MicroPrep columns (Zymo Research), or from BG3 cells using Zymo ZR RNA MiniPrep columns (Zymo Research). Twelve to fifteen wing imaginal discs were used per biological replicate, and at least 3 biological replicates were generated per genotype.
|
| Label |
biotin
|
| Label protocol |
Biotin-labeled cRNA was synthesized from 200 ng of starting RNA and using Affymetrix GeneChip 3′ IVT Express kit according to the manufacturer's instructions.
|
| |
|
| Hybridization protocol |
Fragmented RNA was hybridized to each Affymetrix (Santa Clara, CA) GeneChip Drosophila Genome 2.0 array. The arrays were washed and stained on the GeneChip Fluidics Station 450 series.
|
| Scan protocol |
Arrays were scanned on an Affymetrix GeneChip® Scanner 3000 7G.
|
| Description |
Gene expression data from Ph-depleted BG3 cells
|
| Data processing |
Probe cell intensities for each array were normalized using GCRMA algorithms, which consist of background adjustment and quantile normalization, accounting for probe GC content. Normalization was executed using the R statistical environment [R Foundation for Statistical Computing, Vienna, 2007; ISBN 3-900051-07-0; www.R-project.org] and the Bioconductor package (www.bioconductor.org).
|
| |
|
| Submission date |
Nov 07, 2012 |
| Last update date |
Apr 25, 2013 |
| Contact name |
Cheri A Schaaf |
| E-mail(s) |
cheri.schaaf@gmail.com
|
| Phone |
314-977-9224
|
| Organization name |
Saint Louis University School of Medicine
|
| Department |
Biochemistry & Molecular Biology
|
| Lab |
Dorsett
|
| Street address |
1100 South Grand Ave
|
| City |
St Louis |
| State/province |
MO |
| ZIP/Postal code |
63104 |
| Country |
USA |
| |
|
| Platform ID |
GPL1322 |
| Series (2) |
| GSE42105 |
Cohesin and Polycomb proteins functionally interact to control transcription at silenced, restrained, and active genes [expression array data] |
| GSE42106 |
Cohesin and Polycomb proteins functionally interact to control transcription at silenced, restrained, and active genes |
|
