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Sample GSM5620874

Query DataSets for GSM5620874

Status

Public on Oct 13, 2021

Title

Ethanol-treated_sample_rep1

Sample type

SRA

Source name

Ethanol-treated_sample

Organism

Drosophila melanogaster

Characteristics

strain: Canton-S
developmental stage: Larvae (third instar)
treatment: Ethanol (grown in 5% ethanol disloved in regular food (v/v))
tissue: dissected larval brains (pool of ~30 brains)

Treatment protocol

Chronic alcohol exposure was performed following the procedure described by Robinson et al. (2012a). Briefly, standard cornmeal food was supplemented with pure alcohol (Ethyl alcohol no. S73985A, Fisher Scientific) to obtain a 5% alcohol concentration in food (alcohol food). After melting the standard fly food for approximately 30 seconds, 2.65 mL of 95% alcohol was added to 50 mL of fly food just before re-solidification. Food was stirred to homogenize the solution and allowed to cool at room temperature for 24 hours. After the 24 hours were through, adult CS files were transferred to each bottle and allowed to lay eggs for 24 hours. At the end of the 24 hours, adult flies were discarded. The alcohol food was supplemented daily by pipetting 0.3 mL with 10% alcohol on the surface of the food until larvae reached the third instar stage, ~5-6 days after eggs had been laid. For control larvae, the food was supplemented with water when melted instead of alcohol (control food). Control food was not supplemented daily.

Growth protocol

All flies were maintained on a standard cornmeal/molasses/agar medium under 12:12 light:dark conditions

Extracted molecule

polyA RNA

Extraction protocol

Six days after egg-laying, as larvae began to crawl out of the food to proceed with pupae formation, individual third-instar larvae were collected using a needle, transferred to microcentrifuge tubes, and frozen at -80°C for 24 to 48 hours. Freezing was followed by the addition of 200uL of RNAlater-ICE Frozen Tissue Transition Solution (AM7030, Thermo Fisher Scientific) to larvae. Approximately 30 larval brains were dissected per replicate in RNAlater-ICE, for a total of three replicates (~90 larval brains) for each group. Total RNA was extracted from larval brains using EZ1 RNA Tissue mini kit (EZ1 RNA Tissue Mini Kit no. 959034, Qiagen). All samples were treated with DNase I (DNase Set no. 79254, Qiagen). RNA concentration and quality were evaluated using a Qubit 2.0 fluorometer (Life Technologies, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA).
Capture of mRNA (poly-A selection) and library preparation was carried out using the Illumina TruSeq® Stranded mRNA Library Prep kit from 300ng of total RNA. Libraries were sequenced with an Illumina NextSeq 500/550 High Output Kit v2.5 for 80 cycles following manufacturer’s protocol (Illumina, Inc). At least 20 million reads were obtained for each replicate (a pool of 30 brains).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

The generated sequencing data was stored in FASTQ format and, the quality of the sequencing reads was assessed using FastQC 0.11.9.
Adapters present in the sequences were eliminated using Trimmomatic 0.39.
Tools from the “new tuxedo suite” were used to process the reads following the protocol by Pertea et al. (2016). Briefly, sequence alignment to the Drosophila dm6 genome assembly was completed using HISAT 2.2.0; SamTools was used to compress, sort, and index transcripts, while merging and quantifying transcripts was done using StringTie 2.1.4.
Calculation of differential expression was achieved using Ballgown (Pertea et al., 2016)
Genome_build: dm6
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each transcript in each Sample
Supplementary_files_format_and_content: tab-delimited text files with differential expression values (fold change, p-value, q-value) between Control and Ethanol treated samples calcualated for each transcript

Submission date

Oct 10, 2021

Last update date

Oct 13, 2021

Contact name

Alfredo Ghezzi

E-mail(s)

[email protected]

Organization name

University of Puerto Rico, Rio Piedras

Department

Department of Biology