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Sample GSM1258038

Query DataSets for GSM1258038

Status

Public on Nov 04, 2013

Title

Dpse_ovary_rep1

Sample type

SRA

Source name

ovary

Organism

Drosophila pseudoobscura

Characteristics

strain: Drosophila Species Stock Center 14011-0121.94
genotype: wild type
developmental stage: 6-10 day old adult
tissue: ovary

Growth protocol

All flies were grown on cornmeal-molasses agar at 20C on 12 hour light/dark cycle at ~50% humidity.

Extracted molecule

polyA RNA

Extraction protocol

Virgin adult males and females were collected and aged 6-10 days before dissecting out testes or ovaries. All attached ducts were removed from the gonad tissue. Tissues were stored in RNAlater (Ambion) at -20C until RNA extraction. Immediately before RNA extraction RNAlater was removed by adding 1 mL RNase-free phosphate-buffered saline (PBS), centrifuging the sample 5,000 x g at 4 degrees C for 5 minutes, replacing the supernatant with 1 mL fresh PBS, centrifuging again, and replacing the supernatant with extraction buffer (XB) from the Arturus(R) PicoPure (R) kit according to the manufacturer's instructions. We included an on-column DNase digestion in the RNA extraction protocol.
Libraries were constructed following the Illumina TruSeq RNA Library Construction Kit. Poly-A+ RNA was selected, reverse-transcribed using random (hexamer) priming, and the resulting cDNA fragments sheared to ~120-200 bp fragments. These fragments were used for adapter ligation and sequencing.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer IIx

Data processing

genome_build: Drosophila pseudoobscura release 3.1 (http://flybase.org)
Raw data processing: Illumina's Real Time Analysis v 1.13 software was used to process sequencer images, call bases, and provide base quality scores
Mapping reads to reference genomes: Bowtie v2.1.0 using default parameters and the appropriate reference genome.
Differential expression analysis: Cufflinks v 2.1.0 Cuffdiff module, default parameters.
Differential expression analysis: edgeR. Count values for genes were generated using HTseq v0.5.4p3 with options "-s no -m intersection-nonempty -a 1". Count values were full-quantile normalized within samples by gene-level GC content calculated from reference genome/annotation files and full-quantile normalized between samples before DE analysis.
Supplementary_files_format_and_content: Cuffdiff files are native Cuffdiff v2.1.0 gene_exp.diff output files. In each case, sample_1 is testis and sample_2 is ovary. Value_1 and value_2 are then the normalized expression values (FPKM) of the locus that were used in the DE analysis. Please see the software manual for additional information on the file format.
Supplementary_files_format_and_content: edgeR files are native output from edgeR's "topTags" subroutine. Information for all genes that were tested is included in this file, which consists of columns for gene ID, log2(normalized ovary expression level / normalized testis expression level), logCPM, likelihood ratios, P-values, and q-values. See the software manual for further information.

Submission date

Nov 04, 2013

Last update date

May 15, 2019

Contact name

Maria D Vibranovski

E-mail(s)

mdv@ib.usp.br

Organization name

Universidade de Sao Paolo

Department

Genetics and Evolutionary Biology