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| Status |
Public on Aug 25, 2017 |
| Title |
trr_ChIPseq_wildtype_rep2 |
| Sample type |
SRA |
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| Source name |
Drosophila heads
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: head
developmental stage: adult (0-5 days old)
genotype: Wild type
chip antibody: anti_Trr (Johnston et al., 2011)
extraction protocol: Chromatin was extracted from 50 µl aliquots of frozen wildtype fly heads, aged between 0 and 5 days old, in biological duplicates. Fly heads were crushed in PBS (Sigma) and crosslinked with formaldehyde (Sigma) at a final concentration of 1% for 30 minutes. Crosslinking was terminated using glycine (Invitrogen) at a final concentration of 125mM and crosslinked material was immediately washed twice in PBS and centrifuged at 13000rpm, for 15 minutes at 4 degrees. The pellet was resuspended in buffer 1 (15 mM Tris-Hcl (pH 7.5), 60 mM KCl, 15 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 0.1 mM sucrose) and the solution was homogenized using a QiaShredder column (Qiagen). Cells were lysed using buffer 1 supplemented by 2% triton-X-100 (Sigma) and a crude nuclear extract was collected by centrifugation (6000 rpm, 10 minutes at 4 degrees). Nuclei were re-suspended in incubation buffer (0.15% SDS, 1% triton x-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris) supplemented with 0.1% BSA (Sigma) and 1x protease inhibitor (Roche) and subjected to sonication (Diagenode Bioruptor) for 30 minutes (30 seconds on/off cycle using the “high intensity” mode), yielding average DNA fragments of 150-300 base pairs. Immunoprecipitation reactions were performed overnight in incubation buffer with 3 ng of anti-trr antibody (gift from Dr. A. Mazo), protease inhibitor cocktail (Roche), BSA, and pre-blocked protein A/G agarose beads (Santa Cruz). Chromatin-antibody-bead complexes were recovered by centrifugation (4000rpm, 2 minutes at four degrees) and washed twice with low salt buffer (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris pH 8, 150 mM NaCl), once with high salt buffer (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris pH 8, 500 mM NaCl), once with LiCl wash buffer (10 mM Tris pH 8.0, 1% Na-deoxycholate, 1% NP-40, 250 mM LiCl, 1 mM EDTA) and twice with TE buffer. Chromatin was eluted in 1% SDS, 0.1M NaHCO3, 200 mM NaCl and decroslinked at 65° Celcius (C) for four hours. DNA was purified by phenol/chloroform extraction and ethanol precipitated using linear acrylamide (Ambion) and sodium acetate at -20 degrees. Library preparation for Illumina sequencing was performed using the Truseq DNA sample prep kit V2 (Illumina) with approximately 3 ng of starting DNA 15 PCR cycles for amplification. Library fragment size was assessed using the 2100 Agilent Bioanalyser and was shown to be between 200 and 400 bp.
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| Treatment protocol |
Wild type, UAS/Gal4 mediated knock down of Trr and G9a mutant Drosophila melanogaster heads
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| Growth protocol |
Flies were cultured according to standard methods at 25 degrees with a 12 hour light dark cycle
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq libraries were prepared for sequencing using standard Illumina protocols (Trueseq)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
none
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| Data processing |
Quality control: fastqc and Htseq_qa.py
ChIP-seq: Reads were mapped to the Drosophila genome (BDGP R5/dm3 using BWA 0.6.1 with standard settings allowing 1 mismatch. Total alignment efficiency was more than 95%. Duplicate reads and reads with a mapping quality score (MAPQ) below 15 were excluded from downstream analysis. For each biological replicate, trr binding sites were identified using MACS2 with input DNA as control (MACS2 for trr_ChIP_seq_wildtype_rep1: band width=300, mfold: 5,50, p-value: 1*10^-3, regional labda: 1000-10000. For trr_ChIPseq_wildtype_rep2: band width: 300, mfold: 3,100, q-value: 1*10^-5, lambda: 500-5000). Bindings sites on chromosome U, Uextra and mitochondrial genome were not used for further analysis. Remaining putative trr binding regions were visualized in a heatmap that was sorted based on k-means clustering of read intensity around the centre of the peak using the python script fluff_heatmap.py (https://github.com/simonvh/fluff). Individual clusters were examined visually in the genome browser to assess the quality of the peaks within the clusters. Two clusters (8 and 15) of replicate 1 were identified that contained many false positive peaks with a relatively small binding region and cluster 15 had very few reads around the centre. Peaks within these clusters were removed from downstream analysis. For trr_rep2 all clusters appeared to represent high quality trr binding sites. Trr binding regions from both biological replicates were merged and concatenated and the number of reads present in these regions were counted in each sample individually using HTSeq. and normalized to library size. The ratio and mean of reads in each binding region was calculated between the two biological duplicates to identify trr binding sites that are consistent between the two biological replicates. We included all binding sites with a mean number of reads > 100 and < 2 fold difference in normalized read count between the two biological replicates.
genome build: BDGP R5/dm3
processed data files format and content: wig file
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| Submission date |
Nov 02, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Tom Koemans |
| E-mail(s) |
tom.koemans@radboudumc.nl
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| Organization name |
Radboudumc
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| Department |
Human genetics
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| Lab |
van Bokhoven/ Schenck
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| Street address |
Geert Grooteplein 10
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| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
Netherlands |
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| Platform ID |
GPL13304 |
| Series (1) |
| GSE89459 |
Genome wide binding of trr (ChIP-seq) and expression analysis (RNA-seq) of trr- and G9a mutant fly heads |
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| Relations |
| BioSample |
SAMN05968765 |
| SRA |
SRX2321784 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2372604_ChIP_trr2.wig.gz |
8.7 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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