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Sample GSM5542901

Query DataSets for GSM5542901

Status

Public on Feb 27, 2022

Title

MA6-Cd45-BRU4: Pooled Male FHM Control Brain RepD

Sample type

SRA

Source name

Brain tissue

Organism

Pimephales promelas

Characteristics

Sex: Male
tissue: Brain
treatment: Negative control
replicate: D
exposure time: 96 hours

Treatment protocol

Fathead minnows were exposed for 96 hours

Growth protocol

Adult fathead minnows were exposed to graded aqueous fluoxetine concentrations (2.42, 10.7, 56.7 µg/L) in 20L tanks for 96 hours. Two males and three females were in each tank. Tanks were in a flow-through design maintained at 25°C and a 16:8h light:dark cycle

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from tissue samples of individual fish using the RNeasy Plus Universal Mini Kit (Qiagen, Hilden, Germany) in a QiaCube instrument (Qiagen). Pooled samples with an RNA Integrity Number (RIN) ≥ 9 were used for mRNA sequencing
Steps for generating libraries from total RNA included the following: mRNA enrichment was performed using the NEBNext Poly(A) Magnetic Isolation Module (New England BioLabs, Ipswich, MA, USA); cDNA synthesis was done using NEBNext RNA First Strand Synthesis and NEBNext Ultra Directional RNA Second Strand Synthesis Modules (New England BioLabs). Subsequent steps of library preparation were done using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs). Adapters and PCR primers were purchased from New England BioLabs. Libraries were quantified using the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (Kapa Biosystems, Wilmington, MA, USA). Determination of average fragment size was done with a LabChip GX (PerkinElmer, Waltham, MA, USA) instrument. Once libraries were normalized and pooled, they denatured in 0.02N NaOH and neutralized using HT1 buffer. The pool was loaded at 225pM on an Illumina NovaSeq (Illumina, San Diego, CA, USA) S4 lane using Xp protocol. The run was performed for 2x100 cycles (paired-end mode). A phiX library was used as a control and mixed with libraries at 1% level. Base calling was performed with RTA v3.4.4. Program bcl2fastq2 v2.20 was applied to demultiplex samples and generate fastq reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

https://galaxy.ecotoxxplorer.ca/
Trim Galore! -- trimmed to a minimum phred score of 20 and a minimum sequence length of 35 bases per paired read
Kallisto quant -- alignment
Count data was normalized by use of the trimmed mean of M values approach (Robinson and Oshlack, 2010)
Genome_build: Fathead minnow reference transcriptome NCBI Acc# GCA_016745375.1
Supplementary_files_format_and_content: csv -- Differential expression results using DESeq2

Submission date

Aug 27, 2021

Last update date

Feb 27, 2022

Contact name

Markus Hecker

E-mail(s)

carly.colville@usask.ca, markus.hecker@usask.ca

Organization name

University of Saskatchewan

Street address

44 Campus Drive