|
| Status |
Public on Dec 31, 2017 |
| Title |
SMR wing disc rep1 |
| Sample type |
genomic |
| |
|
| Source name |
SMR wing disc replicate 1
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: L3 imaginal wing disc
antibody: Abcam rabbit anti-GFP (ab290)
|
| Treatment protocol |
N/A
|
| Growth protocol |
For each ChIP experiment we used 50 Drosophila wing imaginal discs harvested from L3 wanering larvae. 3 independent ChIP samples were pulled for each biological replicate
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
30 third instar wandering transgenic larvae carrying the target fusion protein transgene (SMRTER-YFP) were dissected in 1 x PBS (Phosphate-Buffered Saline). The dissected larval heads were inverted to expose the wing discs. Fat was removed from the larval heads when possible. The inverted larval heads were then transferred to a 1.5ml tube and were fixed with 1ml of 4% PFA (+1.5mM of EGS for the SMRTER-YFP genotype) at room temperature for 25 minutes. Following fixation, 200μl of 2M-glycine solution was added to quench the reaction. The larval heads were then rinsed 3 times with cold PBS, after which they were incubated in 1 x PBS + Roche cOmplete protease inhibitor cocktail. Wing discs were then removed from the larval head carcasses and once 50 wing discs were collected they were centrifuged at 5000rpm at room temperature for 1 minute. After spinning down the discs, the supernatant was carefully removed and replaced with 50μl of nuclear lysis buffer (50 mM Tris-HCl pH 8; 10 mM EDTA; 1% SDS; 1x Roche cOmplete protease inhibitor). The sample was then incubated on ice for 10 minutes after which the wing discs were homogenised with a disposable plastic pestle. 250μl of IPDB (ImmunoPrecipitation Dilution Buffer) was then added to the mixture. The sample was sonicated for 7 minutes with a 30 second “ON/OFF” cycle setting in a pre-cooled bioruptor (Diagenode UCD-200). After sonication, the sample was centrifuged at 13200rpm at 4oC for 10 minutes, after which the chromatin containing supernatant was transferred to a new 1.5ml tube and was further diluted with 200μl of IPDB.
|
| Label |
biotin
|
| Label protocol |
Library was generated using GenomePlex® Complete Whole Genome Amplification (WGA) Kit #WGA2-50RXN. The amplification reaction was complemented by adding 0.75ul of 10mM dUTP. The amplification was performed for 14 cycles. Samples were then fragmented and hybridized to GeneChip Drosophila Tiling 2.0R Array according to Affymetrix specifications.
|
| |
|
| Hybridization protocol |
Samples were prepared and hybridized to GeneChip Drosophila Tiling 2.0R Array according to Affymetrix specifications.
|
| Scan protocol |
The tiling arrays were scanned using an Affymetrix GeneChip Scanner 3000 7G according to manufacturer's instruction.
|
| Data processing |
The CEL files were processed with Biconductor Starr and Ringo packages, signal values were quantile normaised between all samples and the normalised log2 rations of sample/input reported for each replicate experiment as *.sgr files (chromosome, start, ratio).
WD_Smr_EGS_r1.sm_quant-all_Ringo_hw300v8.sgr.gz file contains the median of replicate ratios smoothed into window scores using a sliding window of 600bp and 10 minimum Probes in window with the Bioconductor Ringo package. The bed file was created assuming a normalNull distribution centred around zero and the resulting p-values were adjusted for multiple testing using the fdr method.
|
| |
|
| Submission date |
Apr 10, 2017 |
| Last update date |
Dec 31, 2017 |
| Contact name |
Sarah Bray |
| E-mail(s) |
sjb32@cam.ac.uk
|
| Organization name |
University of Cambridge
|
| Department |
PDN
|
| Street address |
Downing Street
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 3DY |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL6629 |
| Series (2) |
| GSE97602 |
Genome -wide profile of SMRTER binding in Drosophila wing imaginal discs |
| GSE97603 |
Genome-wide profile of Notch pathway co-repressors and their role in shaping the Notch response |
|
