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| Status |
Public on Sep 22, 2016 |
| Title |
Sample_test_0_gpp_rep1 |
| Sample type |
SRA |
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| Source name |
Testes
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| Organism |
Drosophila melanogaster |
| Characteristics |
Sex: male
heat_hours: 0
genotype: w* / Y; +; P{tubP-GAL4}LL7, P{tubP-GAL80ts}7 / P{TRiP.HMS00160}attP2
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| Growth protocol |
All Drosophila strains were maintained at The University of Geneva. We reared the flies on Drosophila food (per liter: 6.2 g agar, 59 g cornmeal, 59 g yeast, 5 mL propionic acid and 26 mL 99% methyl 4-hydroxybenzoate). Homozygous w*; +; P{tubP-GAL4}LL7, P{tubP-GAL80ts}7 virgin females were crossed to homozygous males carrying a UAS responsive transgene or homozygous males of control lines at 20 oC, and the progeny were reared at that temperature. Adult progeny were collected at 0-4 hours post-eclosion and were placed into fresh vials and shifted to 29 oC as indicated.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Flies were removed from the indicated incubation temperatures and dissected immediately in Phosphate Buffered Saline solution at room temperature. Each sample was a pool from three flies. The testes were removed at the junction with the vas deferens. Ovaries were removed at the common oviduct, and any eggs in the ovipositor were also removed. Each dissected carcass or gonad was immediately crushed into 100 μL RNAlater solution (Life Technologies, Carlsbad CA) and sealed in a deep-well 96-well plate at room temperature after dissection and during transportation. After the samples arrived at NIH they were stored at -80 oC. For RNA extraction, 20-30 glass beads (1.0 mm dia, BioSpec Products, Bartlesville OK) were added to each well of samples with 100 μL frozen RNAlater. Then we added 600 μL RLT buffer from the RNeasy 96 kit (Qiagen, Valencia CA) to each well. After all samples were completed thawed, we homogenized the samples three times for 60 seconds each with a mini-beadbeater (Biospec Products, Bartlesville OK). Extraction of total RNA was performed using the RNeasy 96 kit according to manufacturer's guide (RNeasy 96 Protocol for Isolation of Total RNA from Animal Cells, III Using spin technology centrifuge), except that we used 700 μL of 70% ethanol (Warner-Graham, Cockeysville MD) in each well to dilute the RNAlater and RLT buffer. We mixed 400 ng of total RNA in 50 μl of nuclease-free water (Quality Biological, Gaithersburg MD). Then we prepared the polyA+ RNA with Dynabeads Oligo(dT)25 (Life Technologies, Carlsbad CA) and followed a strand-specific library preparation pipeline (Lee et al., Plos Genetics, 2016, PMID 27599372). We added to each sample 20 pg of External RNA Control Consortium (ERCC) spike-ins (Lee et al., Journal of Genomics, 2016, PMID 27512518) before the mRNA fragmentation step. In the first plate with all samples from the restrictive temperature experiment, ERCC pool 78A was added for all, except the sham control samples, for which we added ERCC pool 78B. In the second plate with all samples from the permissive temperature experiment, ERCC pool 78B was added for all, except sham control samples, for which we added ERCC pool 78A. Libraries were quantified with Quant-iT PicoGreen (Life Technologies, Carlsbad CA), and pooled for multiplexed sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
We performed single-end 50 bp sequencing on the HiSeq2000 Sequencing System (Illumina, San Diego CA) according to the manufacturer. De-multiplexed reads (Chastity > 0.6) FASTQ output was from Illumina CASAVA v.1.8.2. Reads were mapped to FlyBase release 6 version 4 of the Drosophila melanogaster genome appended with the ERCC sequences (Jiang et al., Genome Research, 2011, PMID 21816910; errors in the original beta-ERCC annotation were corrected: the ERCC-00009, ERCC-00014, ERCC-00057, ERCC-00059, ERCC-00099, ERCC-00108, ERCC-00116 were strand-corrected, and ERCC-00085 was identified as Subpool C). We used STAR (v2.4.2a) (Doblin et al., Bioinformatics, 2012, PMID 23104886) with pre-build annotation (--runMode genomeGenerate). We used HTSeq (v0.6.1p1) (--stranded=reverse) (Anders et al., 2015, Bioinformatics, PMID 25260700) to calculate gene-level read counts, and SAMtools (v0.1.19) (Li et al., Bioinformatics, 2009, PMID 19505943) to index chromosomes and sort alignments.
Genome_build: BDGP release 6 (obtained from FlyBase (http://www.flybase.org)
Supplementary_files_format_and_content: For this sample, there is a tab-delimited text file that contains HTSeq-generated read counts for each gene. There are also two supplementary files for the whole GSE77492 datasets: a tab-delimited file GSE77492_merged.star_htseq_read_count.txt that contains HTSeq-generated read counts for each gene and sample, and a GSE77492_dmel-all-r6.04.ERCC.gtf file that contains the annotation of Drosophila melanogaster (FlyBase release 6 version 4) plus the ERCC spike-ins.
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| Submission date |
May 04, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Brian Oliver |
| E-mail(s) |
[email protected]
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| Phone |
301-204-9463
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| Organization name |
NIDDK, NIH
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| Department |
LBG
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| Lab |
Developmental Genomics
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| Street address |
50 South Drive
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL13304 |
| Series (1) |
| GSE77492 |
Gene expression following: RNAi knockdown of grappa, lilliputian, or Suppressor of Triplolethal; induction of Cyclin-dependent kinase 9 dominant negative constructs, or ectopic expression of stand still in Drosophila melanogaster. |
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| Relations |
| Reanalyzed by |
GSM3278995 |
| BioSample |
SAMN04942752 |
| SRA |
SRX1742670 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2143095_Sample_test_0_gpp_rep1.star_htseq_read_count.txt.gz |
64.4 Kb |
(ftp)(http) |
TXT |
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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