Truseq mrna

Manufactured by Illumina

Sourced in United States

The TruSeq mRNA is a lab equipment product designed for RNA sequencing. It is used for the preparation of mRNA samples for sequencing on Illumina platforms.

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9 protocols using truseq mrna

RNA-seq Library Preparation and Alignment

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RNA extraction was performed using the AllPrep DNA/RNA Mini Kit (Qiagen, Venlo, Germany). Libraries for RNA next-generation sequencing were generated by TruSeq mRNA (Illumina) and sequenced on an Illumina NovaSeq 6000 with 2 × 150 bp and 30 Mio read pairs per sample. Reads were aligned to the human genome (Homo sapiens GRCh38) using STAR software v. 2.6 [12 (link),13 (link)].
Mapped reads were counted with HTSeq [13 (link)]. Read counts were normalized with DESeq2 for sequencing depth and RNA composition using the median of ratios method. [14 (link)].
A complete analysis of transcriptomic data will be published elsewhere (manuscript in preparation).

Hoppe S., Meder L., Gebauer F., Ullrich R.T., Zander T., Hillmer A.M., Buettner R., Plum P., Puppe J., Malter W, & Quaas A. (2022). Trophoblast Cell Surface Antigen 2 (TROP2) as a Predictive Bio-Marker for the Therapeutic Efficacy of Sacituzumab Govitecan in Adenocarcinoma of the Esophagus. Cancers, 14(19), 4789.

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Venom Transcriptomes of Parasitic Wasps

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Venom transcriptomes were generated for each species by pooling 50 venom apparatuses (venom gland + reservoir). Following dissections, samples were immediately collected into 40μl of 4°C TRIzol Reagent^® (Invitrogen) in 1.5μl microcentrifuge tubes and stored at −80°C. Six whole bodies of adult females were also collected from U. rufipes and T. sarcophagae to acquire a more complete gene set. Total RNA extractions were performed as per the TRIzol Reagent^® manufacturer’s protocol, followed by quantification and quality checking using Agilent 2100 Bioanalyzer. TruSeq mRNA (Illumina) library construction and 100bp paired-end sequencing on Illumina HiSeq 2500 platform were performed by University of Rochester Genomics Research Center (URGRC). The starting material for all the venom libraries was normalized to 1ng RNA. cDNA for each species was indexed with a unique adapter. Each library was normalized by equimolar multiplexing before sequencing at ~ 1 library/10^th of a lane. In total, for this project venom gland transcriptomes were sequenced for U. rufipes, T. sarcophagae, N. vitripennis, and N. giraulti and whole body adult female transcriptomes were sequenced for U. rufipes and T. sarcophagae.

Martinson E.O., M.r.i.n.a.l.i.n.i., Kelkar Y.D., Chang C.H, & Werren J.H. (2017). The evolution of venom by co-option of single copy genes. Current biology : CB, 27(13), 2007-2013.e8.

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Strand-specific mRNA-Seq Library Preparation

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Strand-specific paired-end reads with a fragment length of 100 bp for each sample were generated by Edinburgh Genomics, using the Illumina TruSeq mRNA library preparation protocol (poly-A selected; Illumina; Part: 15031047 Revision E). mRNA-Seq libraries were sequenced on an Illumina NovaSeq 6000 platform to generate >66 M paired-end reads per sample (min: 6.6e+07, max: 1.21e+08, mean: 9.17+e07).

Salavati M., Woolley S.A., Cortés Araya Y., Halstead M.M., Stenhouse C., Johnsson M., Ashworth C.J., Archibald A.L., Donadeu F.X., Hassan M.A, & Clark E.L. (2021). Profiling of open chromatin in developing pig (Sus scrofa) muscle to identify regulatory regions. G3: Genes|Genomes|Genetics, 12(2), jkab424.

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Comparative Evaluation of Illumina RNA-Seq Kits

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Three kits from Illumina were tested with different total RNA input: TruSeq mRNA with an input amount of 1 μg, TruSeq stranded mRNA with inputs of 1 μg and 100 ng and TruSeq stranded total RNA Gold with 1 μg and 100 ng. The TruSeq mRNA and stranded mRNA kits use polyA selection to trap the mRNA before proceeding to the cDNA synthesis. In the case of the TruSeq stranded total RNA Gold kit, the RiboZero Gold technology targets the rRNA with baits, and the cDNA synthesis takes place after purification. To ensure that the information on the strand is kept in the experimental procedure, the stranded kits use both actynomycin-D (during the first strand cDNA synthesis) and dUTP (during the second strand synthesis). The libraries are end-repaired and adenylated before being ligated with Y-shape single indexed adaptors, and then are amplified by PCR. We followed the Illumina recommendations for all the kits, except for the last purification step which we performed with Ampure XP beads at 0.8X to remove all the adapter dimers.

Palomares M.A., Dalmasso C., Bonnet E., Derbois C., Brohard-Julien S., Ambroise C., Battail C., Deleuze J.F, & Olaso R. (2019). Systematic analysis of TruSeq, SMARTer and SMARTer Ultra-Low RNA-seq kits for standard, low and ultra-low quantity samples. Scientific Reports, 9, 7550.

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Transcriptome Profiling of Chicken RNA-seq

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Library preparation for RNA sequencing (RNAseq) was also performed by Edinburgh Genomics using the Illumina TruSeq mRNA (poly-A selected) library preparation protocol. mRNA was sequenced at a depth of >40 million strand-specific 75 bp paired end reads per sample, using an Illumina HiSeq 4000. Expression was quantified using the high speed quantification tool Kallisto v0.43.1 (Bray et al., 2016 (link)) following procedures detailed previously (Bush et al., 2017 (link); Bush et al., 2018 (link)). Kallisto quantifies expression at the transcript level by building an index of k-mers from a set of reference transcripts and then mapping the RNA-seq reads to it, matching k-mers generated from the reads with the k-mers present in the index. Transcript-level estimates (transcripts per million, TPM) are then summarised to the gene level. The current analysis used the revised chicken reference transcriptome defined previously (Bush et al., 2018 (link)).

Freem L., Summers K.M., Gheyas A.A., Psifidi A., Boulton K., MacCallum A., Harne R., O’Dell J., Bush S.J, & Hume D.A. (2019). Analysis of the Progeny of Sibling Matings Reveals Regulatory Variation Impacting the Transcriptome of Immune Cells in Commercial Chickens. Frontiers in Genetics, 10, 1032.

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RNA-seq analysis of neurodegenerative models

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For the RNA-seq of the mouse samples, total RNA was extracted from MACS-isolated microglia of each models using an RNeasy Mini Kit (Qiagen, Hilden, Germany). The RNAs were sampled from cerebral cortices of 8-month-old App^{NL-G-F/NL-G-F} and 7-month-old rTg4510 mice and lumbar spinal cords of 5-month-old SOD^G93A mice together with the corresponding wild-type or control mice, respectively. For the RNA-seq of the human samples, total RNA was prepared from precuneus of frozen postmortem brain using mirVana™ miRNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer instructions. The total RNA was qualified by using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Libraries were prepared by using TruSeq mRNA or TruSeq Stranded mRNA (Illumina, San Diego, CA, USA), and, from these libraries, 151-nt paired-end reads were sequenced on the HiSeq X Ten with the HiSeq X Reagent Kits (Illumina) and the NovaSeq 6000 with the NovaSeq Reagent Kits (Illumina).

Sobue A., Komine O., Hara Y., Endo F., Mizoguchi H., Watanabe S., Murayama S., Saito T., Saido T.C., Sahara N., Higuchi M., Ogi T, & Yamanaka K. (2021). Microglial gene signature reveals loss of homeostatic microglia associated with neurodegeneration of Alzheimer’s disease. Acta Neuropathologica Communications, 9, 1.

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RNA-seq analysis of plant stress response

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For RNA-seq library preparation, total RNA was treated with Ambion Turbo DNase (Thermo Fisher Scientific, AM1907) and quantified using the Qubit RNA BR Assay Kit (Invitrogen, Q10210). Four biological replicates (n = 4) were used for 0-min samples of Col-0, camta3-1, fer-4, and piezo and 22-min samples of Col-0 and piezo. Three biological replicates (n = 3) were used for 22-min samples of camta3-1 and fer-4. Then, 500 ng of RNA was used for library preparation using Illumina TruSeq mRNA (poly-A selection) and TruSeq RNA UD Indexes for up to 96 samples (Illumina, 20022371). Samples were pooled and sequenced on a half Illumina NovaSeq6000 S4 lane, 2 × 150 bp reads, incl Xp kit. Data were processed using demultiplexing and quality controlled with FastQC. Alignment of reads was performed against the TAIR11 annotation using STAR (67 (link)). On average, 32 million reads per sample were generated. Counts were assigned to genes using featureCounts (68 (link)), and analysis of DEGs was performed with DeSEQ2 (69 (link)). Transcripts were considered differentially expressed if padj < 0.05 and fold change ≥ 1.5 or fold change ≤ −1.5. Raw RNA-seq data files are available on ArrayExpress (accession number E-MTAB- 10920). GO enrichment analysis was performed using TF2Network and GOrilla (70 (link)).

Darwish E., Ghosh R., Ontiveros-Cisneros A., Tran H.C., Petersson M., De Milde L., Broda M., Goossens A., Van Moerkercke A., Khan K, & Van Aken O. (2022). Touch signaling and thigmomorphogenesis are regulated by complementary CAMTA3- and JA-dependent pathways. Science Advances, 8(20), eabm2091.

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RNA Extraction and Sequencing from Skeletal Muscle

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RNA extraction from skeletal muscle tissue was performed using the phenol based TRIzol method (Invitrogen #15596018, Thermo Fisher Scientific, Waltham, MA, USA), quantified and quality checked using the 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA, USA). Libraries were prepared by poly-A selection (TruSeq mRNA, Illumina, San Diego, CA, USA) and multiplexed at the National Genomics Infrastructure Sweden. Clustering was done by ‘cBot’ and samples were sequenced on NovaSeq6000 (NovaSeq Control Software 1.6.0/RTA v3.4.4) with two lanes of a 2x151 setup ‘NovaSeqXp’ workflow in ‘S4’ mode flow cell. The Bcl to FastQ conversion was performed using bcl2fastq_v2.20.0.422 from the CASAVA software suite. The quality scale used is Sanger/phred33/Illumina 1.8+. QC and processing were performed using the nfcore/rnaseq analysis pipeline publicly available at github (https://github.com/nf-core/rnaseq). The RNA-sequencing data has been deposited at the European Genome-phenome Archive (EGA) which is hosted at the EBI and the CRG, under accession number EGA: EGAS00001006139.

Reitzner S.M., Emanuelsson E.B., Arif M., Kaczkowski B., Kwon A.T., Mardinoglu A., Arner E., Chapman M.A, & Sundberg C.J. (2023). Molecular profiling of high-level athlete skeletal muscle after acute endurance or resistance exercise – A systems biology approach. Molecular Metabolism, 79, 101857.

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Transcriptomic Profiling of Glioblastoma Subgroups

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Next generation sequencing and gene expression microarray experiments were performed at the Genomics and Bioinformatics Core Facility at the Norwegian Radium Hospital, Oslo University Hospital (Norway). The library preparation for RNA sequencing was performed using the Truseq mRNA Illumina protocol, and the samples were sequenced on the Illumina HiSeq platform (paired end 2 × 75 bp). Normalized expression data was further analyzed in J-Express 2011. Subgrouping of the GSC cultures as proneural or mesenchymal was performed by analyzing gene expression microarray data using the HumanHT-12 chip (Illumina). Unsupervised hierarchical clustering was performed according to the gene panels described by Mao et al. and Phillips et al. [24 (link), 25 (link)]. Quality issues led to one culture (T1461) not being successfully sequenced and could not be included in the gene expression analyses.

Skaga E., Kulesskiy E., Fayzullin A., Sandberg C.J., Potdar S., Kyttälä A., Langmoen I.A., Laakso A., Gaál-Paavola E., Perola M., Wennerberg K, & Vik-Mo E.O. (2019). Intertumoral heterogeneity in patient-specific drug sensitivities in treatment-naïve glioblastoma. BMC Cancer, 19, 628.

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