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Truseq stranded mrna reagents

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The TruSeq Stranded mRNA reagents are a library preparation kit designed for sequencing of mRNA transcripts. The kit provides a method for converting mRNA into a library of cDNA fragments with adapter sequences, which can then be amplified and sequenced.

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Lab products found in correlation

Hiseq 2500, by Illumina (8 mentions) Fragment analyzer, by Agilent Technologies (8 mentions) Sciclone liquid handling robot, by PerkinElmer (7 mentions) Truseq sr cluster kit v4 reagents, by Illumina (6 mentions) Truseq sbs kit v4 reagents, by Illumina (3 mentions) Hiseq 3000 4000 sbs kit reagents, by Illumina (3 mentions) Bcl2fastq2 conversion software, by Illumina (3 mentions) Trizol reagent, by Thermo Fisher Scientific (3 mentions) Bcl2fastq conversion software v2, by Illumina (3 mentions) Spin column based rna purification kit, by Macherey-Nagel (2 mentions) Hiseq 3000 4000 sr cluster kit reagents, by Illumina (2 mentions) Hiseq sr cluster kit v4 reagents, by Illumina (2 mentions) Hiseq sbs kit v4 reagents, by Illumina (2 mentions) Hiseq 4000, by Illumina (2 mentions) Syber green qpcr, by Thermo Fisher Scientific (1 mentions) Superscript 2, by Thermo Fisher Scientific (1 mentions) Anti cd3 and anti cd28 antibody, by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Rnaeasy mini kit, by Qiagen (1 mentions) Sybr green qpcr, by Thermo Fisher Scientific (1 mentions)

17 protocols using truseq stranded mrna reagents

1

Transcriptomic Analysis of Primary Brain Endothelial Cells

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Ch25hfl/fl and Ch25hECKO female mice were injected with tamoxifen. Nine brains per genotype were pooled and primary brain microvascular endothelial cells (pMBMEC) were isolated and plated in a 96‐well plate (2 wells/brain). Confluent pMBMEC were left unstimulated or stimulated IL‐1β for 24 h. RNA of three wells was pooled to obtain one replicate for RNA sequencing. The Lausanne Genomic Technologies Facility performed the RNA‐seq. RNA quality was assessed on a Fragment Analyzer (Agilent Technologies), and all RNAs had a RQN between 8.7 and 10. RNA‐seq libraries were prepared from 500 ng of total RNA with the Illumina TruSeq Stranded mRNA reagents (Illumina) using a unique dual indexing strategy, and following the official protocol automated on a Sciclone liquid handling robot (PerkinElmer). Libraries were quantified by a fluorometric method (QubIT, Life Technologies) and their quality assessed on a Fragment Analyzer (Agilent Technologies).
Cluster generation was performed with 2 nM of an equimolar pool from the resulting libraries using the Illumina HiSeq 3000/4000 SR Cluster Kit reagents and sequenced on the Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents for 150 cycles (single end). Sequencing data were demultiplexed using the bcl2fastq2 Conversion Software (version 2.20, Illumina).
Ruiz F., Peter B., Rebeaud J., Vigne S., Bressoud V., Roumain M., Wyss T., Yersin Y., Wagner I., Kreutzfeldt M., Pimentel Mendes M., Kowalski C., Boivin G., Roth L., Schwaninger M., Merkler D., Muccioli G.G., Hugues S., Petrova T.V, & Pot C. (2023). Endothelial cell‐derived oxysterol ablation attenuates experimental autoimmune encephalomyelitis. EMBO Reports, 24(3), e55328.
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2

Transcriptome Analysis of DNMT and TET Knockouts

Cited 1 time
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Total RNA was extracted and DNAse-I treated using a spin column-based RNA purification kit (Macherey–Nagel). Reverse transcription was performed with 500 ng of RNA using random primers and SuperScriptII (Invitrogen). Primers were designed using Primer 3 [84 (link)] and used for SYBER Green qPCR (Applied Biosystems). All primers sequences are listed in Additional file 9: Table S1. For mRNA sequencing, 100-bp RNA-seq libraries were prepared using 200 ng of total RNA and the Illumina TruSeq Stranded mRNA reagents (Illumina). Three replicates from independent experiments for each sample for wild-type and DNMT TKO cells and two replicates for TET TKO cells were selected for high-throughput sequencing. Cluster generation was performed with the resulting libraries using the Illumina TruSeq SR Cluster Kit v4 reagents and sequenced on an Illumina HiSeq 2500 (Illumina).
Coluccio A., Ecco G., Duc J., Offner S., Turelli P, & Trono D. (2018). Individual retrotransposon integrants are differentially controlled by KZFP/KAP1-dependent histone methylation, DNA methylation and TET-mediated hydroxymethylation in naïve embryonic stem cells. Epigenetics & Chromatin, 11, 7.
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3

RNA-seq analysis of STING-deficient T cells

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For RNA sequencing naïve T cells were sorted from the spleens of wild-type mice and STINGgt/gt mice and expanded in the presence of anti-CD3 and anti-CD28 antibodies (eBioscience) for 2 days. Cells were rested 1 day in RPMI-1640 medium supplemented with 10% (v/v) FCS, 5% HEPES buffer (Amimed), 2% L-glutamine (Life Technologies) and Ciprofloxacin (Bayer Schering Pharma), without antibodies and stimulated with either DMSO or 125 μg/ml CMA overnight. Total RNA was isolated from the cells using the RNAeasy Mini Kit (Qiagen). RNA-seq libraries were prepared using 1000 ng of total RNA and the Illumina TruSeq Stranded mRNA reagents (Illumina) on a Sciclone liquid handling robot (PerkinElmer) using a PerkinElmer-developed automated script. Cluster generation was performed with the resulting libraries using the Illumina TruSeq SR Cluster Kit v4 reagents and sequenced on the Illumina HiSeq 2500 using TruSeq SBS Kit v4 reagents. Sequencing data were processed using the Illumina Pipeline Software version 1.82. Heat maps were produced from normalised expression data using Cluster 3.0 for computation and JTreeview for visualisation.
Gulen M.F., Koch U., Haag S.M., Schuler F., Apetoh L., Villunger A., Radtke F, & Ablasser A. (2017). Signalling strength determines proapoptotic functions of STING. Nature Communications, 8, 427.
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4

RNA-seq Analysis of Mouse Transcriptome

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Total RNA was extracted and DNAse-I treated using a spin column-based RNA purification kit (Macherey-Nagel). cDNA was prepared with SuperScript II reverse transcriptase (Invitrogen). Primers (listed in Supplemental Experimental Procedures) were used for SYBR Green qPCR (Applied Biosystems), and specificity was confirmed with dissociation curves. For mRNA sequencing, 100-bp single-end RNA-seq libraries were prepared using the Illumina TruSeq Stranded mRNA reagents (Illumina). Cluster generation was performed with the resulting libraries using the Illumina TruSeq SR Cluster Kit v4 reagents. Sequencing was performed with Illumina HiSeq 2500 in 100-bp reads run. The RNA-seq reads were mapped to the mm9 genome using TopHat (Kim et al., 2013 (link)), allowing multimapped reads to be randomly assigned once among the mapped loci. Gene counts were generated with HTseq-count program using default parameters, and TE counts were computed using BEDtools (multicov). Sequencing depth normalization and differential expression analyses were performed using the voom function of Bioconductor package LIMMA (Law et al., 2014 (link)).
Ecco G., Cassano M., Kauzlaric A., Duc J., Coluccio A., Offner S., Imbeault M., Rowe H.M., Turelli P, & Trono D. (2016). Transposable elements and their KRAB-ZFP controllers regulate gene expression in adult tissues. Developmental cell, 36(6), 611-623.
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5

Automated RNA-seq Library Preparation

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RNA‐seq libraries were prepared as described in Jan et al (2019). In brief, RNA quality was assessed on a Fragment Analyzer (Advanced Analytical Technologies, Inc., Ankeny, IA, USA). RNA‐seq libraries were prepared using 1,000 ng of total RNA and the Illumina TruSeq Stranded mRNA reagents (Illumina; San Diego, California, USA) on a Sciclone liquid handling robot (PerkinElmer; Waltham, Massachusetts, USA) using a PerkinElmer‐developed automated script. Cluster generation was performed with the resulting libraries using the Illumina TruSeq SR Cluster Kit v4 reagents and sequenced on the Illumina HiSeq 2500 using TruSeq SBS Kit v4 reagents. Sequencing data were processed using the Illumina Pipeline Software version 2.20.
Fujita S., De Bellis D., Edel K.H., Köster P., Andersen T.G., Schmid‐Siegert E., Dénervaud Tendon V., Pfister A., Marhavý P., Ursache R., Doblas V.G., Barberon M., Daraspe J., Creff A., Ingram G., Kudla J, & Geldner N. (2020). SCHENGEN receptor module drives localized ROS production and lignification in plant roots. The EMBO Journal, 39(9), e103894.
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6

Transcriptome sequencing from total RNA

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RNA extraction was performed using TRIzol reagent (Thermo Fisher Scientific) followed by DNase treatment and clean up with RNeasy MinElute Cleanup kit (Qiagen), according to the manufacturer’s instructions. RNA quality was assessed on a Fragment Analyzer (Agilent Technologies) and all RNAs had an RQN between 9.8 and 10. RNA-seq libraries were prepared using 500 ng of total RNA with the Illumina TruSeq Stranded mRNA reagents (Illumina Technologies) following the manufacturer’s recommendations. The poly-A RNA was selected, the RNA was cleaved and converted to cDNA, the fragments were end-repaired and ligated to the adapters, and the cDNA libraries were amplified by PCR. Libraries were quantified by a fluorimetric method and their quality assessed on a Fragment Analyzer. Cluster generation was performed from the resulting libraries using the Illumina HiSeq SR Cluster Kit v4 reagents and sequenced on the Illumina HiSeq 2500 using HiSeq SBS Kit v4 reagents for 125 cycles.
Di-Battista A., Favilla B.P., Zamariolli M., Nunes N., Defelicibus A., Armelin-Correa L., da Silva I.T., Reymond A., Moyses-Oliveira M, & Melaragno M.I. (2023). Premature ovarian insufficiency is associated with global alterations in the regulatory landscape and gene expression in balanced X-autosome translocations. Epigenetics & Chromatin, 16, 19.
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7

Assessing RNA Quality and Performing RNA-seq

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RNA quality was assessed on a Fragment Analyzer (Advanced Analytical Technologies, Inc., Ankeny, IA, USA) and all RNAs had a RQN between 9.4 and 10.
RNA-seq libraries were prepared using 500 ng of total RNA and the Illumina TruSeq Stranded mRNA reagents (Illumina; San Diego, California, USA). Cluster generation was performed with the resulting libraries using the Illumina TruSeq SR Cluster Kit v4 reagents and sequenced on the Illumina HiSeq 2500. Sequencing data were demultiplexed using the bcl2fastq Conversion Software (v. 2.20, Illumina; San Diego, California, USA).
Hendrikx S., Coso S., Prat-Luri B., Wetterwald L., Sabine A., Franco C.A., Nassiri S., Zangger N., Gerhardt H., Delorenzi M, & Petrova T.V. (2019). Endothelial Calcineurin Signaling Restrains Metastatic Outgrowth by Regulating Bmp2. Cell reports, 26(5).
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8

Transcriptome Analysis of E. coli Strains

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RNA was isolated using SV Total RNA isolation system (Promega) from MG1655 and IF72 cells grown exponentially (OD ~0.15) in GluCAA medium. The isolated RNA was analyzed on an Agilent Bioanalyzer to confirm quality and depleted for rRNA using Ribo-Zero rRNA Removal Kit for Gram-Negative Bacteria (Illumina).
The rRNA depleted samples were submitted to the Norwegian Sequencing Centre (sequencing.uio.no) where libraries were prepared using TruSeq stranded mRNA reagents (Illumina) according to manufacturer’s instructions, entering the procedure at the RNA fragmentation step with 35 ng rRNA-depleted RNA, and fragmenting for 4 minutes at 94°C. Libraries were sequenced on an Illumina NextSeq-500 instrument with 150 cycle mid-output v1 reagents, according to manufacturer's instructions, employing 75 bp paired-end reads. Image analysis and base calling were performed using Illumina's RTA software version 2.1.3. Reads were filtered to remove those with low base call quality using Illumina's default chastity criteria.
The sequencing was performed on three replicates from each of the two strains.
Flåtten I., Fossum-Raunehaug S., Taipale R., Martinsen S, & Skarstad K. (2015). The DnaA Protein Is Not the Limiting Factor for Initiation of Replication in Escherichia coli. PLoS Genetics, 11(6), e1005276.
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9

RNA-seq Library Preparation for Splenic and Bone Marrow NK Cells

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Total RNA was extracted from sorted cells using the RNeasy plus mini kit (Qiagen) according to the manufacturer's instructions. Clontech RNA-seq libraries were prepared for the splenic NK cell preparations. Double stranded cDNA for RNA-seq library preparation was generated using SMART-Seq v4 Ultra Low Input RNA reagents (Catalog Number 634888, Clontech) according to the protocol provided with the reagents beginning with 10 ng of total RNA and using 9 cycles of PCR. 150 pg of the resulting cDNA were used for library preparation with the Illumina Nextera XT DNA Library reagents (Catalog Number 15032354, Illumina) using the single cell RNA-seq library preparation protocol developed for the Fluidigm C1 (Fluidigm).
TruSeq stranded mRNA-seq libraries were prepared for the bone marrow-derived NK cells. RNA-seq libraries were prepared using 400 ng of total RNA and the Illumina TruSeq Stranded mRNA reagents (Illumina) according to the protocol supplied with the reagents and starting with 100ng of total RNA on a Sciclone liquid handling robot (PerkinElmer) using a PerkinElmer-developed automated script.
Library sequencing cluster generation was performed with the resulting libraries using the Illumina TruSeq SR Cluster Kit v4 reagents and sequenced on the Illumina HiSeq 2500 using TruSeq SBS Kit v4 reagents. Sequencing data were processed using the Illumina Pipeline Software version 1.82.
Castro W., Chelbi S.T., Niogret C., Ramon-Barros C., Welten S.P., Osterheld K., Wang H., Rota G., Morgado L., Vivier E., Raeber M.E., Boyman O., Delorenzi M., Barras D., Ho P.C., Oxenius A, & Guarda G. (2018). The transcription factor Rfx7 limits metabolism of NK cells and promotes their maintenance and immunity. Nature immunology, 19(8), 809-820.
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10

RNA Extraction and RNA-Seq Analysis in Mice Kidneys

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RNA from frozen half-kidneys of 72 mice were extracted and purified using RNAeasy MiniElute Spin Column (Qiagen). RNA quality was assessed on a Fragment Analyzer (Agilent Technologies). All RNAs had an RNA quality number (RQN) between 7.5 and 9.7. RNA-Seq libraries were prepared from 200 ng of total RNA with the Illumina TruSeq Stranded mRNA reagents (Illumina) using a unique dual indexing strategy and following the official protocol automated on a Sciclone liquid handling robot (PerkinElmer). Libraries were quantified by a fluorometric method (QubIT, Life Technologies) and their quality assessed on a Fragment Analyzer (Agilent Technologies). Clusters were generated with 2 nM of an equimolar pool from the resulting libraries using the Illumina HiSeq 3000/4000 SR Cluster Kit reagents. Sequencing was performed on the Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents for 150 cycles (single read). Sequencing data were demultiplexed, filtered for failed reads, and written to FASTQ files using the bcl2fastq2 conversion software (version 2.20, Illumina). Details for RNA-Seq reads mapping are described in Supplemental Methods.
Bignon Y., Wigger L., Ansermet C., Weger B.D., Lagarrigue S., Centeno G., Durussel F., Götz L., Ibberson M., Pradervand S., Quadroni M., Weger M., Amati F., Gachon F, & Firsov D. (2023). Multiomics reveals multilevel control of renal and systemic metabolism by the renal tubular circadian clock. The Journal of Clinical Investigation, 133(8), e167133.
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