Transcript profiling was conducted in the liver of 6 females per treatment group. For the MG treatment group, only 3 individuals were analysed because there were only three females in the replicate tanks for this treatment. RNA was extracted from female livers using an RNeasy Mini extraction kit (Qiagen), incorporating on-column DNase treatment, according to the manufacturer’s instructions. The concentration, purity and integrity of RNA were determined using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., USA). All RNA input to library construction was of high quality with A260/A280 and A260/A230 ratios > 1.8 and RIN scores > 8. ERCC spike-in control mixes (Ambion) were added to all individual RNA samples, according to the manufacturer’s instructions to allow for analysis of the accuracy of the transcript quantification and dynamic range. cDNA libraries from all samples were then prepared using the Illumina TruSeq RNA Sample Preparation kit, multiplexed with 24 samples per lane and sequenced using an Illumina HiSeq 2500, to generate 100 bp paired reads.
Ercc spike in control mixes
Manufactured by Thermo Fisher Scientific
Sourced in United States
ERCC spike-in control mixes are a set of synthetic RNA transcripts designed to serve as external reference controls for RNA-sequencing experiments. The mixes contain a defined set of RNA sequences at known concentrations, which can be used to assess the performance and sensitivity of RNA-sequencing workflows.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Hiseq 2500, by Illumina (3 mentions)
Truseq rna sample preparation kit, by Illumina (2 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rneasy mini extraction kit, by Qiagen (1 mentions)
Rneasy mini extraction columns, by Qiagen (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Truseq stranded mrna library prep kit, by Illumina (1 mentions)
Allprep dna rna micro kit, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer instrument, by Agilent Technologies (1 mentions)
Hiseq 2500 sequencing system, by Illumina (1 mentions)
Agilent rna 6000 nano kit, by Agilent Technologies (1 mentions)
On column dnase 1 digestion, by Qiagen (1 mentions)
Neb poly a mrna magnetic isolation module, by New England Biolabs (1 mentions)
Truseq small rna sample preparation kit, by Illumina (1 mentions)
Agencourt ampure xp system, by Beckman Coulter (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Rna 6000 nano kit, by Agilent Technologies (1 mentions)
Epicentre scriptseq v 2 rna seq library preparation kit, by Illumina (1 mentions)
5 protocols using ercc spike in control mixes
1
Liver Transcriptome Profiling with ERCC Controls
2
Liver Transcriptome Profiling Protocol
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Transcript profiling was conducted in the livers of three sexually mature males per treatment group. RNA was extracted from livers with TRI reagent (Sigma-Aldrich) according to the manufacturer's instructions and then further purified and treated with DNase on RNeasy Mini extraction columns (Qiagen). The concentration, purity, and integrity of RNA were determined using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies) and an Agilent 2100 Bioanalyzer (Agilent Technologies). All RNA input to library construction was of high quality with 260/280 and 260/230 ratios >1.8 and RNA integrity number scores >8. External RNA Controls Consortium (ERCC) spike-in control mixes (Ambion) were added to all individual RNA samples, according to the manufacturer's instructions. cDNA libraries from all 15 samples were then prepared using the Illumina TruSeq RNA Sample Preparation kit, multiplexed with 24 samples per lane (together with samples from another project) and sequenced using an Illumina HiSeq 2500 to generate 100 bp paired-end reads, according to the manufacturer's instructions.
3
Brain RNA Isolation and Sequencing
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Total RNA was isolated from the whole brain samples using the AllPrep DNA/RNA Micro Kit (Qiagen, Venlo, Holland) incorporating on-column DNase treatment, according to the manufacturer’s instructions. RNA samples were assessed for quality and purity using spectrophotometry and an Agilent 2100 Bioanalyzer Instrument (Agilent Technologies, Santa Clara, CA, USA) in conjunction with the Agilent RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. The RNA integrity numbers (RIN) ranged from 8.30 to 10.00 (Supplementary Table 1 ).
ERCC spike-in control mixes (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) were added to all individual RNA samples, according to the manufacturer’s instructions. cDNA libraries from all samples were then prepared using the TruSeq Stranded mRNA Library Prep kit (Illumina, San Diego, CA, USA), multiplexed with eight samples per lane and RNA-sequencing was carried out on an Illumina HiSeq 2500 Sequencing System (Illumina, San Diego, CA, USA) to generate 100 bp paired reads.
ERCC spike-in control mixes (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) were added to all individual RNA samples, according to the manufacturer’s instructions. cDNA libraries from all samples were then prepared using the TruSeq Stranded mRNA Library Prep kit (Illumina, San Diego, CA, USA), multiplexed with eight samples per lane and RNA-sequencing was carried out on an Illumina HiSeq 2500 Sequencing System (Illumina, San Diego, CA, USA) to generate 100 bp paired reads.
4
Transcriptome and Small RNA Analysis of Atlantic Salmon
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Raw reads were processed using cutadapt v1.4.1 [33 ] to remove adapters and low quality reads. These sequences were aligned against External RNA Controls Consortium (ERCC Spike-In Control Mixes, Ambion) using bowtie2 v2.2.3 [34 (link)] for library prep quality control. After quality control, cleaned RNA-seq data were aligned using TopHat2 v2.0.11 [35 (link)] using Atlantic salmon genome and its reference gene annotation GTF [36 (link)]. Reads mapped to each transcript were counted using featureCounts v1.4.6. [37 (link)]. Gene ontology (GO) terms were assigned by blasting the annotated transcripts to GO database [38 (link)]. Enrichment analysis was performed using topGO an R package [39 ] using differential accumulated transcripts GO terms and all annotated GO terms as a background. Fisher exact test was used, and p-value < 0.01 was considered as a significance threshold.
Small RNA analysis was performed after trimming adapter sequences using cutadapt [33 ]. miRNAs were annotated using miRDeep2 [40 (link)] using Atlantic salmon miRNAs from miRBase21 [41 (link)].
Small RNA analysis was performed after trimming adapter sequences using cutadapt [33 ]. miRNAs were annotated using miRDeep2 [40 (link)] using Atlantic salmon miRNAs from miRBase21 [41 (link)].
5
Transcriptomic Profiling of Gill Tissues
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RNA was extracted from gill tissues (n = 61, n = 4 or 5 per treatment time point and n = 4 for 0-hour time point) using the miRNeasy mini kit (Qiagen) with an on-column DNase I digestion (Qiagen). RNA integrity was assessed using the RNA 6000 Nano kit on the Agilent 2100 Bioanalyzer (Agilent). For mRNA sequencing (RNA-seq) RNA was spiked with External RNA Controls Consortium (ERCC) spike-in control mixes (Ambion) and mRNAs were purified using the NEB Poly(A)mRNA magnetic isolation module (NEB). Libraries were constructed using the Epicentre ScriptSeq v2 RNA-seq library preparation kit (Illumina) with Epicentre ScriptSeq Index PCR primers and the Agencourt AMPure XP system (Beckman Coulter) to purify cDNA. Libraries were pooled and sequenced to 100 bp in paired end mode on the Illumina HiSeq 2500. miRNA sequencing libraries (miRNA-seq) were produced using the gel-based Tru-Seq small RNA sample preparation kit (Illumina) with indexed adapters 1 – 36. Libraries were combined into two pools and sequenced across two lanes to 50 bp in single end mode on the Illumina HiSeq 2500.
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