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| Status |
Public on Aug 02, 2021 |
| Title |
TRAP control rep1 |
| Sample type |
SRA |
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| Source name |
dopaminergic neurons
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain: w1118XdTH-Gal4
tissue: brain
age: post natal day 30
genotype: dTH+TRAP(GFP-RpL10a)
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| Treatment protocol |
For trap experiments, both male and female flies were used. In each experiment, the dTH-Gal4 heterozygous flies were used as control. For the TRAP analysis of fly DA neurons, translated mRNAs in DA neurons were purified from dTH-GAL4/UAS-GFP::RpL10A with and without hPARIS wt or mutant (C571A)
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| Growth protocol |
Human PARIS and C571A constructs cloned and expressed in pUAST plasmid (DGRC) were microinjected into w1118 embryos (The BestGene, Inc). Transgenic dTH-Gal4 lines were obtained from the Bloomington Drosophila Stock Center (BDSC). All experimental maintenance and crosses between transgenic lines were made at 25 °C. Drosophila melanogaster fly stocks were handled using standard protocols, maintained in a 12 h light/dark cycle and fed Drosophila standard diet consisting of cornmeal, agar, yeast, sucrose, and dextrose.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The libraries were prepared from three independent biological samples from 500 (control and hPARIS mutant lines) - 1,500 (hPARIS wt line) of a mixture of male and female heads, whereas a sequencing library using the total mRNA from whole head was prepared for one biological sample to provide quality-assurance of DA neuron-specific translatome.
Libraries for RNA sequencing were prepared with the TruSeq RNA Sample preparation v2 kit (Illumina) and sequenced on an Illumina HiSeq 2000 sequencer.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
GeneCountMatrix_CNT_WB.txt
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| Data processing |
Illumina Casava1.7 software used for basecalling.
The TRAP-seq dataset composed of a total of 10 fastq files representing 4 different treatment groups was processed using the “alternate” differential expression workflow, a modified version of the “new” Tuxedo pipeline at http://ccb.jhu.edu/software/stringtie/index.shtml?t=manual#deseq. Briefly, after quality control (FASTQC v0.11.6) read alignment was performed using HISAT2 v2.1.0, default parameters with “min-intronlen 74” and “max-intronlen 75000”. After multi-mapped reads were identified exclusively specific for rRNA genes, StringTie v2.0’s by-default multi-mapping correction was enabled during transcript abundance estimations and generation of read coverage tables not to compromise the dataset by discarding multi-mapped reads, keeping the sample size (i.e. per-replicate-read-number) above the accepted threshold. StringTie output was directly analyzed for transcript-level differential expression (DE) using the R package Ballgown v2.16.0 while the same output was preprocessed using the author-supplied script “prepDE.py” at the website above for format conversion. These preprocessed files were fed into the R package DESeq2 v1.24.0 for gene-level DE analysis, which ultimately generated the input for the rest of the downstream analysis presented here. The R package NOISeq(sim) v2.28.0 was used for DE analysis of pairwise comparisons with single replicate.
Genome_build: BDGP assembly release 6.28 (Ensembl release 99)
Supplementary_files_format_and_content: Unmapped_hits.bam files: the only use of the unmapped_<...>.bam files provided here is to confirm insertion of the human PARIS gene in the engineered fly genome after a successful TRAP experiment, thereby, reproducing the information presented as Fig. 1d in the manuscript. TopHat v2.0.13 was the choice of aligner with "-G" and "-N 0" arguments to identify unmapped reads in the input fastq files. Bam-to-fastq data conversion before mapping the unmapped reads to the PARIS (ZNF746) DNA sequence (GRCh38) can be made using Hydra v0.5.3, which requires for each input bam file read name sorting by 'samtools sort' with the "-n" argument. If necessary, the repair.sh tool in the BBMap suite can be used to repair the read pairs before alignment. GeneCountMatrix_<...>.txt files: text files containing raw read count information.
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| Submission date |
May 25, 2021 |
| Last update date |
Aug 02, 2021 |
| Contact name |
Karin M Danzer |
| Organization name |
University of Ulm / DZNE, Ulm
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| Department |
Department of Neurology
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| Lab |
Danzer Lab
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| Street address |
University of Ulm Campus, Building N24, R3306 Albert-Einstein-Allee 11
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| City |
Ulm |
| State/province |
Baden-Wurttemberg |
| ZIP/Postal code |
89081 |
| Country |
Germany |
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| Platform ID |
GPL13304 |
| Series (2) |
| GSE175554 |
Integrative genome-wide analysis of dopaminergic neuron-specific PARIS expression dissects recognition of multiple PPAR-γ associated gene regulation (TRAP-Seq). |
| GSE175556 |
Integrative genome-wide analysis of dopaminergic neuron-specific PARIS expression in Drosophila dissects recognition of multiple PPAR-γ associated gene regulation |
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| Relations |
| BioSample |
SAMN19337886 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5341637_unmapped_Sample_dControl_S01_L002.bam |
1.1 Gb |
(ftp)(http) |
BAM |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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