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Sample GSM3912714

Query DataSets for GSM3912714

Status

Public on Jul 23, 2019

Title

Liver, chlorpyrifos-methyl containing diet (Low), Fish16

Sample type

SRA

Source name

Liver

Organism

Gadus morhua

Characteristics

treatment: chlorpyrifos-methyl containing diet (Low)
nominal chlorpyrifos-methyl dose (mg/kg): 0.5
measured chlorpyrifos-methyl dose (mg/kg): 0.5
body weight end (g): 180.93
length end (cm): 27.9
tissue: liver

Treatment protocol

Dietary exposure to chlorpyrifos-methyl containing diet for 30 days

Extracted molecule

total RNA

Extraction protocol

Liver specimen were thoroughly homogenized on dry ice before RNA extraction using the RNeasy Plus mini kit (Qiagen, Oslo, Norway). RNA quantity and quality were determined by using the NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto-CA, USA). The RNA integrity number (RIN value) was 9.3±0.1 (mean±SEM, n=36).
Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s recommendations. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First-Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. After adenylation, NEBNext Adaptor were ligated to prepare for hybridization. Library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA before PCR. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using HiSeq PE Cluster Kit cBot-HS (Illumina) according to the manufacturer’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

18317

Data processing

After cluster generation, the library preparations were sequenced on an Illumina NovaSeq platform and paired-end reads were generated. Clean reads (49,654,219±5,149,769, n=36, mean±st.dev.) were obtained by removing reads containing adapter, reads containing poly-N and low-quality reads from raw data.
Alignment and mapping were conducted by using TopHat2 and the Atlantic cod reference genome (deposited at ENSEMBL, about 70% of clean reads mapped to the genome). RSEM software was used to estimate gene expression levels for each sample (Li et al., 2012). Clean data were mapped back onto the assembled transcriptome, and a read-count for each gene was acquired from the mapping results.
Genome_build: ftp://ftp.ensembl.org/pub/release-81/fasta/gadus_morhua/cds/Gadus_morhua.gadMor1.cds.all.fa.gz
Supplementary_files_format_and_content: Excel file with each line consisting of Atlantic cod ENSEMBL sequence name, and raw counts for all 36 samples

Submission date

Jul 01, 2019

Last update date

Jul 23, 2019

Contact name

Fekadu Yadetie

E-mail(s)

fekadu.yadetie@mbi.uib.no

Organization name

University of Bergen

Department

Biology