|
| Status |
Public on Feb 04, 2018 |
| Title |
brca2[56e/ko] wing disc replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
third instar larval wing discs
|
| Organism |
Drosophila melanogaster |
| Characteristics |
genotype: brca2 mutant
tissue: wing disc
|
| Treatment protocol |
For RNAi treatment of 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 3 hours. For RNAi of all factors, from 0.7 to 40 μg of dsRNA was added per well of a 6-well plate. Media was adjusted to 3 ml and 10% FCS with Schneider'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 each 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.
|
| Growth protocol |
BG3 cells were cultured in Schneider's media with 10% FCS and 10 μg per ml insulin. Drosophila were grown at 25 degrees on cornmeal-molasses-yeast based food.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the Zymo Research Quick-RNA MicroPrep Kits with DNaseI treatment following the manufacturer's directions.
Ion Xpress IonXpressRNA barcoded sequencing libraries were constructed using the Eukaryotic Ribominus and Ion Total v2 kits (Life Technologies) according to the manufacturer's directions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent Proton |
| |
|
| Data processing |
Basecalling was performed by Torrent Suite version 4 and 5 software using the default settings
Alignment to Drosophila melanogaster genome sequence was performed using TMAP map4 algorithm with 5' and 3' softclipping and a minimum seed length of 20 nt
The total sequencing base pair coverage in all exons was summed for all annotated genes in each sample and normalized to total coverage using bedtools and custom R scripts as described in Wu et al. 2015 PLoS Genetics 11:e1005655.
Expression values in RNAi-treated BG3 replicates were averaged and compared to the average of six mock treated BG3 replicates published in Pherson et al. 2017 Sci Adv 3:e1700944 [GSE100548 (GSM2686307, GSM2686308, GSM2686309, GSM2686310, GSM2686311, GSM2686312)]. Expression values in wing discs with mutant brca2 alleles (brca2[56e/ko], brca2[ko]) were averaged and compared to the averaged expression values from wing discs with wild type brca2 alleles (Canton S, y w).
Genome_build: Drosophila melanogaster release 5 (April 2006) with chrU and U_extra sequences removed
Supplementary_files_format_and_content: Microsoft Excel File with normalized sequencing coverage of all annotated genes and statistical comparison of expression in RNAi treated cells to mock RNAi control cells and between wing discs with wild type brca2 to wing discs with mutant brca2 (brca2[56e/ko], brca2[ko]).
|
| |
|
| Submission date |
Jan 18, 2018 |
| Last update date |
Aug 01, 2018 |
| Contact name |
Dale Dorsett |
| Organization name |
Saint Louis University School of Medicine
|
| Department |
Biochemistry and Molecular Biology
|
| Street address |
1100 South Grand Boulevard
|
| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63104 |
| Country |
USA |
| |
|
| Platform ID |
GPL19528 |
| Series (2) |
| GSE109385 |
Roles of the Brca2 and Wapl complexes with Pds5 in sister chromatid cohesion, cohesin localization, and gene expression [RNA-seq] |
| GSE109387 |
Roles of the Brca2 and Wapl complexes with Pds5 in sister chromatid cohesion, cohesin localization, and gene expression |
|
| Relations |
| Reanalyzed by |
GSM3284754 |
| BioSample |
SAMN08377349 |
| SRA |
SRX3586690 |
