Truseq stranded mrna library prep

Manufactured by Illumina

Sourced in United States

The TruSeq Stranded mRNA Library Prep is a laboratory equipment product from Illumina. It is designed to prepare stranded mRNA libraries for sequencing on Illumina platforms. The product includes reagents and protocols to extract, fragment, and reverse transcribe mRNA into cDNA, which is then converted into a sequencing-ready library.

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Lab products found in correlation

Hiseq 4000, by Illumina (15 mentions) Novaseq 6000, by Illumina (12 mentions) Rneasy plus mini kit, by Qiagen (8 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (6 mentions) Rneasy mini kit, by Qiagen (6 mentions) Hiseq 2500, by Illumina (5 mentions) Turbo dna free kit, by Thermo Fisher Scientific (5 mentions) Trizol, by Thermo Fisher Scientific (5 mentions) Qubit rna hs assay kit, by Thermo Fisher Scientific (5 mentions) Kapa library quantification kit, by Roche (4 mentions) Mirneasy mini kit, by Qiagen (4 mentions) Nextseq 500, by Illumina (4 mentions) Tri reagent, by Merck Group (4 mentions) Qubit 4 fluorometer, by Thermo Fisher Scientific (4 mentions) Agilent high sensitivity dna kit, by Agilent Technologies (4 mentions) Tapestation 2200, by Agilent Technologies (3 mentions) 4200 tapestation system, by Agilent Technologies (3 mentions) Ingenuity pathway analysis (ipa), by Qiagen (3 mentions) On column dnase digest, by Qiagen (3 mentions) Tapestation, by Agilent Technologies (3 mentions)

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98 protocols using truseq stranded mrna library prep

RNA-Seq Library Prep for Setd2 Knockdown

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After RNA extraction, RNA integrity was measured with 2200 TapeStation (Agilent) using high sensitivity RNA ScreenTape and reagents (Agilent, 5067–5579, 5067–5580 and 5067–5581). TruSeq Stranded mRNA Library Prep (Illumina, 20020594) and TruSeq RNA Single Indexes Set A (Illumina, 20020492) were used to prepare two Scrambled and two Setd2 knockdown libraries from 900 ng of total RNA per library. Libraries were validated using High Sensitivity D1000 ScreenTape system (Agilent, 5067–5584 and 5067–5585) and KAPA Library Quantification Kit (Kapa Biosystems, 07960140001). All samples were sequenced on one Illumina HiSeq 2500 lane.

Amante S.M., Montibus B., Cowley M., Barkas N., Setiadi J., Saadeh H., Giemza J., Contreras-Castillo S., Fleischanderl K., Schulz R, & Oakey R.J. (2020). Transcription of intragenic CpG islands influences spatiotemporal host gene pre-mRNA processing. Nucleic Acids Research, 48(15), 8349-8359.

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Comprehensive RNA-Seq Library Preparation

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RNAseq was performed at the GeT-PlaGe core facility, INRAE Toulouse. RNA-Seq libraries were prepared using the Illumina TruSeq Stranded mRNA Library Prep, without a poly-A mRNA selection, according to the manufacturer’s protocol. Briefly, RNAs were fragmented to generate double-stranded cDNA and adaptors were ligated. 11 cycles of PCR were applied to amplify the libraries and their quality was assessed using a Fragment Analyser. Libraries were quantified by qPCR using the Kapa Library Quantification Kit (Roche). To reach 5 million bacterial reads for each sample, as recommended in [94 (link)], RNA-Seq reads were generated from three lanes of an Illumina HiSeq3000 and two lanes of an Illumina NOVAseq6000 using a paired-end read length of 2×150 bp. Ribosomal RNA (rRNA) reads were filtered out using sortmerna-2.1b [95 (link)] against Silva and Rfam databases. The raw reads data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB46495 and as specified in S7 Table.

Cohen H., Hoede C., Scharte F., Coluzzi C., Cohen E., Shomer I., Mallet L., Holbert S., Serre R.F., Schiex T., Virlogeux-Payant I., Grassl G.A., Hensel M., Chiapello H, & Gal-Mor O. (2022). Intracellular Salmonella Paratyphi A is motile and differs in the expression of flagella-chemotaxis, SPI-1 and carbon utilization pathways in comparison to intracellular S. Typhimurium. PLoS Pathogens, 18(4), e1010425.

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Transcriptome Analysis of Stricturing CD

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Colonic total RNA samples from two stricturing CD and two non-stricturing CD patients (Cedars-Sinai Medical Center) were used for next-generation whole-transcriptome RNA sequencing (Omega Bioservices). Library was prepared by Illumina TruSeq Stranded mRNA library prep. Sequencing was run on HiSeq 4000/x Ten platform in PE 2x150 format with 5 million reads per sample.

Wang J., Ortiz C., Fontenot L., Xie Y., Ho W., Mattai S.A., Shih D.Q, & Koon H.W. (2020). High circulating elafin levels are associated with Crohn’s disease-associated intestinal strictures. PLoS ONE, 15(4), e0231796.

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Transcriptome Profiling of Cellular RNA

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Total RNA was extracted from cells as described [18 (link)]. RNA concentration was determined with a NanoDropOne spectrophotometer and quality assessed with an Agilent TapeStation4200 (Agilent Technologies, Santa Clara, CA, USA). High-quality RNA from three independent purifications for each experimental point was used for library preparation. Indexed libraries were prepared from 1 μg/ea. of purified RNA with TruSeq Stranded mRNA Library Prep (Illumina, San Diego, CA, USA) following suppliers. Libraries were quantified using the TapeStation 4200 (Agilent Technologies) and Qubit fluorometer (Invitrogen Co., Waltham, MA, USA); then, they were pooled such that each index-tagged sample was present in equimolar amounts. The pooled samples were subjected to cluster generation and sequencing using an Illumina NovaSeq6000 (Illumina) in a 2 × 75 paired-end format. RNA sequencing data have been deposited in the EBI ArrayExpress database (http://www.ebi.ac.uk/arrayexpress, accessed on February 2023) with Accession Number GSE223853.

Accattatis F.M., Caruso A., Carleo A., Del Console P., Gelsomino L., Bonofiglio D., Giordano C., Barone I., Andò S., Bianchi L, & Catalano S. (2023). CEBP-β and PLK1 as Potential Mediators of the Breast Cancer/Obesity Crosstalk: In Vitro and In Silico Analyses. Nutrients, 15(13), 2839.

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Comprehensive Liver RNA Profiling by RNA-Seq

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Total liver RNA was prepared by RNeasy plus mini kit (QIAGEN). The purity and concentration of extracted RNA were measured by 4200 TapeStation system (Agilent). Library prep followed by RNA-sequencing were performed by the National Cancer Institute Sequencing Facility. RNA-seq library was prepared by TruSeq Stranded mRNA Library Prep (Illumina). The library was analyzed by HiSeq3000/4000 system (Illumina) with paired-end 150 read length. The RNA-seq datasets generated during this study are available at Gene Expression Omnibus (Accession number: GSE165521). The comprehensive gene expression profile was subjected to Ingenuity Pathway Analysis (QIAGEN).

Yagai T., Yan T., Luo Y., Takahashi S., Aibara D., Kim D., Brocker C.N., Levi M., Motohashi H, & Gonzalez F.J. (2021). Feedback repression of PPARα signaling by Let-7 microRNA. Cell reports, 36(6), 109506.

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Transcriptome Profiling of Olaparib-Resistant Ovarian Cancer

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RNA was isolated from PEO1 olaparib-sensitive (n=2) and four PEO1 olaparib-resistant clones using RNeasy columns with on-column DNase digest (Qiagen). RNA quality was confirmed using an Agilent Tapestation and all RNA used for library preparation had a RIN>9. Libraries were created using Illumina TruSEQ stranded mRNA library prep (#RS-122-2102). Strand-specific pair-ended libraries were pooled and run on HiSeq4000 (Illumina). Library creation and sequencing were performed at the Genomics Core at the University of Colorado Anschutz Medical Campus. HISAT2 (15 (link)) was used for alignment against GRCh37 version of the human genome. Samples were normalized using TPM (Transcripts per Million) measurement and gene expression using the GRCh37 gene annotation was calculated using home-made scripts. The analysis was performed by the Division of Translational Bioinformatics and Cancer Systems Biology at the University of Colorado School of Medicine. RNA-sequencing has been deposited to NCBI: GSE117765.

Yamamoto T.M., McMellen A., Watson Z.L., Aguilera J., Ferguson R., Nurmemmedov E., Thakar T., Moldovan G.L., Kim H., Cittelly D.M., Joglar A.M., Brennecke E.P., Wilson H., Behbakht K., Sikora M.J, & Bitler B.G. (2019). Activation of Wnt signaling promotes olaparib resistant ovarian cancer. Molecular carcinogenesis, 58(10), 1770-1782.

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RNA-seq of Bap1 in mTSCs

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For RNA-seq, total RNA was extracted with Trizol followed by DNase treatment using TURBO DNA-free kit (Life Technologies AM1907). For wild-type and Bap1 KO mTSC experiments, adapter indexed strand-specific RNA-seq libraries were generated from 1000 ng of total RNA following the dUTP method using the stranded mRNA LT sample kit (Illumina). Libraries were pooled and sequenced on Illumina HiSeq 2500 in 75 bp paired-end mode. FASTQ files were aligned to the Mus musculus GRCm38 genome reference genome using HISAT2 v2.1.0. Sequence data were deposited in ENA under accession ERP023265.
For RNA-seq from SAM Bap1-overexpressing cells, RNA-seq libraries were generated from 500 ng using TruSeq Stranded mRNA library prep (Illumina, 20020594). Indexed libraries were pooled and sequenced on an Illumina HiSeq2500 sequencer in 100 bp single-end mode. FastQ data were map to M. musculus GRCm38 genome assembly using HISAT2 v2.1.0.

Perez-Garcia V., Lea G., Lopez-Jimenez P., Okkenhaug H., Burton G.J., Moffett A., Turco M.Y, & Hemberger M. (2021). BAP1/ASXL complex modulation regulates epithelial-mesenchymal transition during trophoblast differentiation and invasion. eLife, 10, e63254.

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Inducible STAMP Fusion Protein Expression

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For stable cell STAMP fusion protein expression cells were induced with 50ng/ml (low) or 1μg/ml (high) doxycycline in DMEM for 24–72 hours, followed by Trizol extraction and Direct-zol miniprep (Zymo Research) column purification in accordance with manufacturer protocol. Uninduced cells of the same genetic background were used as negative controls. For transient transfections, ~1 million cells were transfected with 2μg expression construct using Fugene HD (Promega) according to manufacterer’s protocol. Upon Agilent TapeStation quantification, 500ng RNA was used as input material to make total RNA-seq libraries with either TruSeq Stranded mRNA Library Prep (Illumina) or KAPA RNA HyperPrep Kit with RiboErase (Roche) following the provided protocols. For mTOR perturbation experiments, cells were treated with 100nM Torin-1 (Cell Signaling) or DMSO vehicle control alongside 1μg/ml doxycycline induction and harvested for RNA after 72 hours 37C incubation.

Brannan K.W., Chaim I.A., Marina R.J., Yee B.A., Kofman E.R., Lorenz D.A., Jagannatha P., Dong K.D., Madrigal A.A., Underwood J.G, & Yeo G.W. (2021). Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes. Nature methods, 18(5), 507-519.

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Bacterial mRNA Enrichment and Depletion

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RNA samples with RNA Integrity Number ≥6.4 were used for the preparation of sequencing libraries. The libraries were constructed using two methods. One was a combination of MICROBExpress Bacterial mRNA Enrichment Kit (Ex) (Thermo Fisher Scientific, Waltham, MA, USA) and TruSeq Stranded mRNA Library Prep (Illumina, San Diego, CA, USA). The other method used the NEBNext rRNA Depletion Kit (Bacteria) (Nx) (New England Biolabs, Inc., Ipswich, MA, USA) and TruSeq Stranded mRNA Library Prep, according to the manufacturer's protocols. An Ex kit was used for the monoculture of each strain and co‐culture of two strains. An Nx kit was used for the monoculture of B. thetaiotaomicron and co‐culture. The final libraries were then sequenced on an Illumina HiSeq 2500 platform with 100 bp paired‐end sequencing reads.

Ikeyama N., Murakami T., Toyoda A., Mori H., Iino T., Ohkuma M, & Sakamoto M. (2020). Microbial interaction between the succinate‐utilizing bacterium Phascolarctobacterium faecium and the gut commensal Bacteroides thetaiotaomicron. MicrobiologyOpen, 9(10), e1111.

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RNA-seq Analysis of Differential Gene Expression

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Total RNA was isolated in three biological replicates. PolyA selection and cDNA library preparation was performed using TruSeq Stranded mRNA Library Prep (Illumina). Paired-end 100 bp sequencing was performed on an Illumina HiSeq 4000 instrument. RNA-seq read files were merged from technical replicates and mapped to the mm10 genome assembly using Tophat (ver 2.0.14) (Trapnell et al., 2009 (link)) with gencode (vM10) annotation used as the transcriptome index. Additional transcripts were assembled using stringtie (1.2.4) (Pertea et al., 2015) and reads within exon sequences counted using HTSeq (HTSeq-0.6.1) counts (Anders et al., 2015 (link)). The differential expression analysis was performed with EdgeR (3.22.3) (using general linear model settings for biological triplicates with blocked matrix model for paired comparisons) (Robinson et al., 2010 (link)). For analysis of Myc targets, the Hallmark Gene Set in the Molecular Signature Database (Broad Institute) (Liberzon et al., 2015 (link)) was used and compared to randomly selected and expression matched genes with statistical significance of differential expression determined with a Kolmogorov-Smirnov test.

Olivero C., Martínez-Terroba E., Zimmer J., Liao C., Tesfaye E., Hooshdaran N., Schofield J., Bendor J., Fang D., Simon M., Zamudio J, & Dimitrova N. (2020). p53 activates the long noncoding RNA Pvt1b to inhibit Myc and suppress tumorigenesis. Molecular cell, 77(4), 761-774.e8.

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