|
Status |
Public on Jan 01, 2017 |
Title |
Body progenitor female biological replicate 1 |
Sample type |
SRA |
|
|
Source name |
Adult Body
|
Organism |
Drosophila melanogaster |
Characteristics |
tissue type: Body genotype: Progenitor Sex: female biological replicate: 1
|
Treatment protocol |
Flies from the progenitor/RNAi OBP56hKK111996 collection from were crossed to a Dll-GAL4 driver (P{w[+mW.hs]=GawB}Dllmd23/CyO). F1 individuals with CyO wings were discarded.
|
Growth protocol |
Flies were reared in vials on cornmeal/molasses/agar medium under standard culture conditions (25°C, 12:12 hour light/dark cycle).
|
Extracted molecule |
total RNA |
Extraction protocol |
We flash froze at -80 °C three-to-five day-old flies, with at least thirty flies per replicate and four replicates per sex per selected population per generation (32 samples) All flies were collected between 8 AM and 11 AM. Total RNA was extracted with Trizol using the RNAeasy Mini Kit (Qiagen, Inc.). rRNA was depleted using the Ribo-Zero™ Gold Kit (Epicentre, Inc.) with 5ug total RNA input. Depleted mRNA was fragmented and converted to first strand cDNA. During the synthesis of second strand cDNA, dUTP instead of dTTP was incorporated to label the second strand cDNA. cDNA from each RNA sample was used to produce barcoded cDNA libraries using NEXTflex™ DNA Barcodes (Bioo Scientific, Inc.) with an Illumina TrueSeq compatible protocol. Library size was selected using Agencourt Ampure XP Beads (Beckman Coulter, Inc.) and centered around 250 bp with the insert size ~130 bp. Second strand DNA was digested with Uracil-DNA Glycosylase before amplification to produce directional cDNA libraries. Libraries were quantified using Qubit dsDNA HS Kits (Life Technologies, Inc.) and Bioanalyzer (Agilent Technologies, Inc.) to calculate molarity. Libraries were then diluted to equal molarity and re-quantified. Two pools of 16 libraries were made, one of the 16 libraries from replicate 1 high and low lines; and one of the 16 libraries from replicate 2 high and low lines. Pooled library samples were quantified again to calculate final molarity and then denatured and diluted to 14pM. Pooled library samples were clustered on Illumina cBot; each pool was sequenced on one lane of Illumina Hiseq2500 using 125 bp single-read v4 chemistry.
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|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
|
|
Data processing |
Demultiplexing: Barcoded sequence reads were demultiplexed using the Illumina pipeline v1.9. Filtering Contaminant Sequences: Adapter sequences were trimmed using cutadapt v1.6 and trimmed sequences shorter than 50bp were discarded from further analysis. Trimmed sequences were then filtered for ribosomal RNA sequences by aligning against a database containing the complete 5S, 18S-5p8S-2S-28S, mt:lrRNA, and mt:srRNA sequences using BWA v0.7.10 (MEM algorithm with parameters ‘-v 2 –t 4’). Alignment: The remaining sequences were aligned to the D. melanogaster genome (BDGP5) and known transcriptome (FlyBase v5.57) using STAR v2.4.0e. Read Counting: Read counts were computed for known gene models using HTSeq-count with the ‘intersection-nonempty’ assignment method. Genome_build: BDGP5/FlyBase5.57 Supplementary_files_format_and_content: Raw read counts for each gene feature in each sample provided as a single table (tab-delimited text). EXPRESSED column indicates whether gene feature met our minimum expression requirements for analysis.
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|
|
Submission date |
Dec 01, 2015 |
Last update date |
May 15, 2019 |
Contact name |
John Shorter |
E-mail(s) |
john_shorter@ncsu.edu
|
Organization name |
North Carolina State University
|
Department |
Biological Sciences
|
Street address |
112 Derieux Place 3510 Thomas Hall
|
City |
Raleigh |
State/province |
NC |
ZIP/Postal code |
27615 |
Country |
USA |
|
|
Platform ID |
GPL17275 |
Series (1) |
GSE75590 |
OBP56h modulates social interactions in Drosophila |
|
Relations |
Reanalyzed by |
GSM3277715 |
BioSample |
SAMN04305405 |
SRA |
SRX1457967 |