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Sample GSM2262766

Query DataSets for GSM2262766

Status

Public on Dec 01, 2016

Title

sim_3L_1

Sample type

SRA

Source name

3rd instar larvae antennal disc

Organism

Drosophila simulans

Characteristics

tissue: Antennal disc
developmental stage: 3rd instar larvae
number of pooled individuals: 70
strain: wild-type

Growth protocol

All flies were reared on cornmeal medium using a 16:8 light:dark cycle at 20°C

Extracted molecule

total RNA

Extraction protocol

Dissected tissue was stored in Trizol reagent for pooling. RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer’s instructions, including an on-column DNase digestion (Qiagen).
RNA concentrations were measured and 700ng RNA was diluted to 60ul total volume with H2O. RNA sequencing libraries were prepared with TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to manufacturer’s instructions. For the RNA fragmentation step, 94˚C, 2min was used with the intention to obtain a median size ~185bp. PCR amplification was done with 15 cycles. A total of 24 multiplexed libraries (barcoded) were accessed for quality and combined together before separation into two identical pooled libraries, which were then subjected to cluster generation followed by Illumina 50bp paired-end sequencing by the UNC High-Throughput Sequencing Facility (HTSF).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

sim_3L_1

Data processing

Basecalls performed using CASAVA version 1.8
Sequenced reads were trimmed for the adaptor sequence, and then mapped to each species’ transcriptome that was downloaded from FlyBase (FB2014_6 release) using bwa-0.7.8
Count tables for each sample were generated using a customized python script, and each transcript ID was given a gene identifier based on ortholog calls from FlyBase. The count tables were then consolidated into matrices containing transcript ID and read counts from all genotypes for each stage using a Ruby script.
Matrices of gene counts were normalized for each species’ developmental time points in R using the DESeq2 suite
Genome_build: FlyBase dsec-r1.3, dere-r1.3, dvir-r1.2, dana-r1.05, dsim-r2.02
Supplementary_files_format_and_content: Comma-separated-values file (.csv) containing normalized transcript counts (baseMean) from DESeq2 for each gene and species for each developmental time point

Submission date

Aug 05, 2016

Last update date

May 15, 2019

Contact name

Jia Wern Pan

Organization name

Duke University

Department

Biology

Lab

Volkan