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| Status |
Public on Mar 17, 2016 |
| Title |
FB.wt_RNASeq_rep2 |
| Sample type |
SRA |
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| Source name |
larval fat bodies
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| Organism |
Drosophila melanogaster |
| Characteristics |
gender: male
genotype: equal mix of the following 6 lines: [1] y,w; M{FL.hsp70P-Dam[4-HT-intein@L127C]}ZH51C [2] y,w; M{FL.hsp70P-Dam[4-HT-intein@L127C]-PC}ZH51C [3] y,w; M{FL.hsp70P-Dam[4-HT-intein@L127C]-H1}ZH51C [4] y,w; M{FL.hsp70P-Dam[4-HT-intein@L127C]-HP1}ZH51C [5] y,w; M{FL.hsp70P-Dam[4-HT-intein@L127C]-MRG15}ZH51C [6] y,w; M{FL.hsp70P-Dam[4-HT-intein@L127C]-BRM}ZH51C
developmental stage: wandering third instar larvae
tissue: fat bodies
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| Treatment protocol |
Fat bodies were dissected from 18 larvae in 1×PBS [140 mM NaCl, 10 mM PO4 Buffer, 3 mM KCl; Invitrogen cat. no. 18912-014] using forceps and dissection needles and collected in TRIzol reagent (Invitrogen, 15596-018) on ice.
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| Growth protocol |
Flies were raised at 25°C on standard cornmeal/molasses/agar medium.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue was homogenized in TRIzol reagent in a 15ml tube using a polytron DI 18 disperser (IKA). Total RNA extraction was performed according to the TRIzol reagent manufacturer’s instructions. Briefly, 0.2× volumes of chloroform (st. amylene) (Biosolve, 030810) was added to the homogenate and the tube was shaken vigorously. The tube was incubated for 3 minutes at room temperature and spun for 1 hour at 4120×g at 4°C. Approximately 70% of the upper aqueous phase was transferred into a new 15ml tube and 0.5× volume of isopropanol (Sigma-Aldrich, 33539) was added and mixed. The tube was incubated for 10 minutes at room temperature and spun for 30 minutes at 4120×g at 4°C. The supernatant was removed and the pellet was washed twice with 80% ethanol (Sigma-Aldrich, 32221). The total RNA pellet was air-dried for 8 minutes, dissolved in an appropriate volume of nuclease free water (Ambion, AM9938) and quantified using a NanoDrop spectrophotometer. The total RNA was further purified using the RNeasy MinElute Cleanup Kit (QIAGEN, 74204) according to the manufacturer’s instructions. Quality (RIN>8) and quantity of the total RNA was assessed by using an Agilent BioAnalyzer 2100 Nano chip (Agilent, 5067-1511).
Illumina TruSeq mRNA libraries were generated using the TruSeq RNA Library Preparation Kit v2 (Illumina, RS-122-2001/2) according to the manufacturer’s instructions. Briefly, polyadenylated RNA from intact total RNA was purified using oligo-dT beads. Following purification, the RNA was fragmented, random primed and reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen, 18064-014). Second strand synthesis was accomplished by using Polymerase I and RNase H. The generated cDNA fragments were 3′ end adenylated and ligated to Illumina paired-end sequencing adapters and subsequently amplified by 15 cycles of PCR. The libraries were analyzed using an Agilent BioAnalyzer 2100 DNA 7500 chip (Agilent, 5067-1506), diluted and pooled equimolar into a 10nM sequencing pool.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
For each replicate, fat bodies were dissected from 3 larvae of each indicated genotype.
processed data file: RNA-seq_Counts.txt, DESeq-processed_FB.control_vs_BR.txt, DESeq-processed_FB.control_vs_FB.25mkM.4HT.txt
FB.control.2
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| Data processing |
Basecalling and filtering were performed using standard software of the Illumina HiSeq 2000.
For each sample, reads were mapped against the Drosophila reference genome (dm3) using TopHat (Trapnell, C. et al. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 25(9), 1105-11, 2009), which was supplied with a known set of gene models. In order to get per sample absolute counts for every gene, HTSeq (Anders, S. et al. HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics 31(2), 166-9, 2015) was used.
Differentially expressed genes were identified by using the R package DESeq (Anders, S. and Huber, W. Differential expression analysis for sequence count data. Genome Biol 11(10), R106, 2010; Anders, S. et al. Count-based differential expression analysis of RNA sequencing data using R and Bioconductor. Nat Protoc 8(9), 1765-86, 2013).
Genome_build: dm3
Supplementary_files_format_and_content: tab-delimited text files
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| Submission date |
Dec 09, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Bas van Steensel |
| E-mail(s) |
[email protected]
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| Phone |
+ 31 20 512 2040
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| Fax |
+31 20 669 1383
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| URL |
http://www.nki.nl/nkidep/vansteensel
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| Organization name |
Netherlands Cancer Institute
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| Department |
division of Molecular Biology
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| Lab |
van Steensel group
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| Street address |
Plesmanlaan 121
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| City |
Amsterdam |
| ZIP/Postal code |
1066 CX |
| Country |
Netherlands |
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| Platform ID |
GPL13304 |
| Series (2) |
| GSE75833 |
Inducible DamID systems for genomic mapping of chromatin proteins in Drosophila [RNA-seq] |
| GSE75835 |
Inducible DamID systems for genomic mapping of chromatin proteins in Drosophila |
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| Relations |
| Reanalyzed by |
GSM3277786 |
| BioSample |
SAMN04327358 |
| SRA |
SRX1472411 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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