|
| Status |
Public on Sep 26, 2017 |
| Title |
Dme_wts_SD_wingdisc_ChIP-nexus 2 |
| Sample type |
SRA |
| |
|
| Source name |
wing discs
|
| Organism |
Drosophila melanogaster |
| Characteristics |
treatment: hand dissections
tissue: wing discs
genotype/variation: y w ubxflp/ y w; FRT82B M(3) ubiGFP/ FRT82B wts149
age: 3rd instar-day9
chip antibody: anti-Sd
|
| Growth protocol |
Normal fly food, 25 degrees.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Third instar larvae were dissected in cold PBS and imaginal disc complexes (anterior one third of the larvae after removing the fat body and salivary glands) were fixed in 1 ml fixation buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.5; 1 mM ethylenediaminetetraacetic acid [EDTA]; 0.5 mM ethylene glycol tetraacetic acid [EGTA]; 100 mM NaCl; 2% formaldehyde) for 30 min at room temperature. Fixed disc complexes were washed 3x fast and 2x 20 minutes with PBST (PBS, pH 7.4; 0.1% Triton X-100; 0.1% Tween-20), and were stored at 4 C until enough discs were obtained. 100-500 wing discs were dissected away from the cuticle and resuspended in buffer A2 (15 mM HEPES, pH 7.5; 140 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 1% Triton X-100; 0.1% sodium deoxycholate; 0.1 % sodium dodecyl sulfate [SDS]; 0.5 % N-lauroylsarcosine; 1 Roche complete protease inhibitor cocktail, cat. no. 5056489001). Tubes were flash frozen in liquid nitrogen and stored at -80 C. Imaginal discs were pooled to reach 500 wt discs (or 100 mutant), and sonication was performed in a Bioruptor sonicator for 5 min (30 s on/off cycle at the high setting) in buffer A2. Following centrifugation (16,000 g; 10 min at 4 C), the supernatant containing soluble chromatin was transferred to fresh tubes, and used for ChIP-nexus.
Library preparation was done as described in He, Johnston and Zeitlinger, 2015 Nature Biotechnology.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Sheared genomic DNA
Custom-made polyclonal rabbit anti-Cic (described in associated paper) and anti-Sd (described in Ikmi et al., 2014, Mol.Biol.Evol.) antibodies were generated and affinity purified by GenScript.
|
| Data processing |
Base calling, read filtering and demultiplexing were performed by Illumina CASAVA 1.8.2 with default settings.
Custom barcode sequences were removed from the first 9bp of each read and sequencing adapters were trimmed from the 3' end using cutadapt v1.3.
Remaining reads with length >= 22bp were aligned to the reference genome using bowtie v1.0.0, retaining only uniquely aligning reads with a maximum of 2 mismatches.
Aligned reads were separated by strand and reduced to a single base at the 5' end.
Genome-wide coverage counts were calculated for each strand separately and saved in BigWig format.
Genome_build: Drosophila melanogaster FlyBase version 5
Supplementary_files_format_and_content: BigWig files contain genome coordinates and counts of the first base of aligned reads, separated by alignment strand.
|
| |
|
| Submission date |
Mar 21, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Jelle Jacobs |
| E-mail(s) |
jelle.jacobs9255@gmail.com
|
| Organization name |
Institute of molecular Pathology
|
| Department |
VBC
|
| Lab |
Stark lab
|
| Street address |
Campus-Vienna-Biocenter 1
|
| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
| |
|
| Platform ID |
GPL19132 |
| Series (1) |
| GSE96868 |
Hippo reprograms the transcriptional response to Ras signalling |
|
| Relations |
| BioSample |
SAMN06624332 |
| SRA |
SRX2659845 |
