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Truseq stranded mrna library prep kit for neoprep

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The TruSeq Stranded mRNA Library Prep Kit for NeoPrep is a reagent kit designed for the preparation of mRNA libraries for sequencing on Illumina's NeoPrep system. The kit enables the generation of stranded mRNA libraries from total RNA samples.

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Lab products found in correlation

Hiseq 4000, by Illumina (3 mentions) Nextseq 550, by Illumina (2 mentions) Hiseq 3000 4000 sbs kit 300 cycles, by Illumina (2 mentions) Hiseq 3000 4000 pe cluster kit, by Illumina (2 mentions) Hiseq 2000, by Illumina (2 mentions) Rneasy mini kit, by Qiagen (2 mentions) Hiseq 2000 platform, by Illumina (2 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions) Nextseq 500 sequencer, by Illumina (1 mentions) Rnaqueous lysis solution, by Thermo Fisher Scientific (1 mentions) Miseq system, by Illumina (1 mentions) Direct zol rna miniprep kit, by Zymo Research (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Superscript 3, by Thermo Fisher Scientific (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Direct zol rna miniprep plus, by Zymo Research (1 mentions) Tri reagent, by Thermo Fisher Scientific (1 mentions) Truseq small rna library prep kit, by Illumina (1 mentions) Real time analysis v2, by Illumina (1 mentions) Bcl2fastq v2, by Illumina (1 mentions)

Spelling variants of this product

TruSeq Stranded mRNA (115 citations) TruSeq Stranded mRNA Library Prep (98 citations) TruSeq kits (44 citations) Stranded mRNA Prep (27 citations) NeoPrep (13 citations) MRNA TruSeq kit (7 citations) TruSeq® Stranded mRNA NeoPrep kit (4 citations)

14 protocols using truseq stranded mrna library prep kit for neoprep

1

Exome and RNA Sequencing of Sarcoma Cell Lines

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Three of the sarcoma cell lines (SA-4, SW872 and LPS510) were exome-sequenced at the Oslo University Hospital Genomics Core Facility using Agilent SureSelectXT Human All Exon v5 protocol and Illumina sequencing by synthesis technology on a HiSeq 4000 instrument (2 × 150 bp). Preprocessing, mapping of the reads and calling of variants was performed as previously described [44 (link)]. RNA-sequencing was performed at the Genomics Core Facility for three of the cell lines, SA-4, SW872 and LPS510, using the Illumina TruSeq Stranded mRNA Library Prep kit for NeoPrep following supplier’s instructions. The libraries were sequenced on a NextSeq 500 Illumina sequencer using a High Output v2 kit chemistry, generating 2 × 75 bp paired-end sequence reads. RNA-Seq reads were aligned using STAR aligner (v.2.5.0b) against the human reference genome (UCSC hg19, RefSeq and Gencode gene annotations), and FPKM estimation for each mRNA was generated by Cufflinks 2 using RNA-seq alignment app at Illumina BaseSpace. The RNA sequencing data for SW872 and SA-4 are available in the Short Read Archive (SRA), with accession number SRP 134156.
Gouravan S., Meza-Zepeda L.A., Myklebost O., Stratford E.W, & Munthe E. (2018). Preclinical Evaluation of Vemurafenib as Therapy for BRAFV600E Mutated Sarcomas. International Journal of Molecular Sciences, 19(4), 969.
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2

RNA-seq of Genetically Modified Cells

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Five hundred thousand cultured cells were harvested, pelleted and lysed in 100 μl of the Ambion RNAqueous lysis solution. RNA was extracted and treated with DNase according to manufacturer’s protocol (RNAqueous®-Micro Kit). mRNA purification, reverse transcription and library preparation were performed using either the automated Neoprep system from Illumina (TruSeq® Stranded mRNA Library Prep Kit for NeoPrep™) or the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs). For each genotype, we sequenced at least two biological replicates except for the CH12 med26 knock-out, for which only a single homozygous knock-out clone was obtained.
Khattabi L.E., Zhao H., Kalchschmidt J., Young N., Jung S., Blerkom P.V., Kieffer-Kwon P., Kieffer-Kwon K.R., Park S., Wang X., Krebs J., Tripathi S., Sakabe N., Sobreira D.R., Huang S.C., Rao S.S., Pruett N., Chauss D., Sadler E., Lopez A., Nóbrega M.A., Aiden E.L., Asturias F.J, & Casellas R. (2019). A pliable Mediator acts as a functional rather than an architectural bridge between promoters and enhancers. Cell, 178(5), 1145-1158.e20.
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3

Illumina-based mRNA Sequencing Analysis

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mRNA libraries were generated using the TruSeq Stranded mRNA Library Prep Kit for NeoPrep (Illumina) and sequenced in pair-end 75 nt mode with a MiSeq system (Illumina). Sequence tags were analyzed using ArrayStudio (OmicSoft). DEGs were extracted with significance at p-values < 0.05 (t-test). The significance of gene set overlapping was assessed with Fisher’s exact test.
Yugami M., Okano H., Nakanishi A, & Yano M. (2020). Analysis of the nucleocytoplasmic shuttling RNA-binding protein HNRNPU using optimized HITS-CLIP method. PLoS ONE, 15(4), e0231450.
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4

RNA-Seq Analysis of HPV18 Genome

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For RNA-Seq, libraries were prepared using Tru-Seq Stranded mRNA Library Prep kit for NeoPrep (Illumina, San Diego, CA, USA) using 100ng total RNA input according to manufacturer’s instructions. Libraries were pooled and run as 75-cycle–pair end reads on a NextSeq 550 (Illumina) using a high-output flow cell. Sequencing reads were aligned to human (GRCh37) and HPV18 (AY262282.1) genomes with STAR aligner (v2.5.2b) [35 (link)]. The computations were performed on the CaStLeS infrastructure [36 ] at the University of Birmingham. Sashimi plots were generated in Integrative Genomics Viewer (IGV), Broad Institute (http://software.broadinstitute.org/software/igv/).
Ferguson J., Campos-León K., Pentland I., Stockton J.D., Günther T., Beggs A.D., Grundhoff A., Roberts S., Noyvert B, & Parish J.L. (2021). The chromatin insulator CTCF regulates HPV18 transcript splicing and differentiation-dependent late gene expression. PLoS Pathogens, 17(11), e1010032.
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5

RNA-seq Library Preparation and Sequencing

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RNA-seq was performed by the next-generation sequencing section at OIST Graduate University. Hundred nanograms of total RNA was used for RNA-seq library preparation with a TruSeq Stranded mRNA Library Prep Kit for NeoPrep (NP-202-1001; Illumina) that allows polyA-oligo(dT)-based purification of mRNA. The manufacturer’s protocol was employed with minor modification and optimization as follows. Custom dual index adapters were ligated at the 5′ and 3′-end of libraries, and PCR was performed for 11 cycles. One hundred fifty-base-pair paired-end read RNA-seq was performed with a Hiseq 3000/4000 PE Cluster Kit (PE-410-1001; Illumina) and a Hiseq 3000/4000 SBS Kit (300 Cycles) (FC-410-1003; Illumina) on a Hiseq4000 (Illumina), according to the manufacturer’s protocol.
Mostafa D., Yanagiya A., Georgiadou E., Wu Y., Stylianides T., Rutter G.A., Suzuki T, & Yamamoto T. (2020). Loss of β-cell identity and diabetic phenotype in mice caused by disruption of CNOT3-dependent mRNA deadenylation. Communications Biology, 3, 476.
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6

Transcriptome Analysis of Floral Buds

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RNA was extracted from unopened floral bud tissue using TRIzol (Thermo Fisher) and the Direct-Zol RNA MiniPrep kit (R2050; Zymo Research) including in-column DNase treatment. Seventy-five nanograms total RNA was used as input for the TruSeq Stranded mRNA Library Prep Kit for Neoprep (NP-202-1001; Illumina). Libraries were sequenced on a HiSeq 2000 (Illumina). Reads were aligned with TopHat, including the fr-firststrand parameter. Cufflinks was used to generate count data using annotation from TAIR10 that was fed into the DEseq2 package in R for differential expression analysis.
Zhang Y., Harris C.J., Liu Q., Liu W., Ausin I., Long Y., Xiao L., Feng L., Chen X., Xie Y., Chen X., Zhan L., Feng S., Li J.J., Wang H., Zhai J, & Jacobsen S.E. (2018). Large-scale comparative epigenomics reveals hierarchical regulation of non-CG methylation in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 115(5), E1069-E1074.
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7

RNA-Seq and qPCR Analysis Protocol

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RNA was extracted with an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol and DNase treated. For RNA-Seq, libraries were prepared using TruSeq Stranded mRNA Library Prep kit for NeoPrep (Illumina, San Diego, CA, USA) using 100 ng total RNA input according to manufacturer’s instructions. Libraries were pooled and run as 75-cycle–pair end reads on a NextSeq 550 (Illumina) using a high-output flow cell.
cDNA was synthesised using Superscript III (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. qPCR was performed using a Stratagene Mx3005P detection system with SyBr Green incorporation and the primers listed in Table 2.
Pentland I., Campos-León K., Cotic M., Davies K.J., Wood C.D., Groves I.J., Burley M., Coleman N., Stockton J.D., Noyvert B., Beggs A.D., West M.J., Roberts S, & Parish J.L. (2018). Disruption of CTCF-YY1–dependent looping of the human papillomavirus genome activates differentiation-induced viral oncogene transcription. PLoS Biology, 16(10), e2005752.
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8

CD8 T Cell Activation Dynamics

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Naïve WT and NfkbiaNES/NES CD8 T cells stimulated with plate bound 5 μg/mL anti-CD3 and 5 μg/mL anti-CD28 antibodies with or without soluble anti-4–1BB antibody for 12 hours. Cells were lysed in TRIzol reagent (Invitrogen) and frozen at −80° C until total RNA was extracted with the Direct-zol RNA MiniPrep Plus (Zymo Research) kit. For RNA-seq, libraries were generated with the TruSeq Stranded mRNA Library Prep Kit for NeoPrep (Illumina) and sequencing was performed on an Illumina HiSeq 2000.
Lisiero D.N., Cheng Z., Tejera M.M., Neldner B.T., Warrick J.W., Wuerzberger-Davis S.M., Hoffmann A., Suresh M, & Miyamoto S. (2020). IκBα nuclear export enables 4–1BB induced cRel activation and IL-2 production to promote CD8 T cell immunity. Journal of immunology (Baltimore, Md. : 1950), 205(6), 1540-1553.
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9

Transcriptomic and Small RNA Profiling of Worms

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Worms were collected in TriReagent (ThermoFisher) and subjected to three freeze-thaw cycles. After phase-separation using 1-bromo-3-chloropropane (BCP), RNA was precipitated in isopropanol at −30°C for 2 h. RNA was pelleted by centrifugation at 21,000 × g for 30 min at 4°C. After two washes in 70% ethanol and one wash in 75% ethanol, the pellet was resuspended in water. mRNA libraries were made using Illumina TruSeq Stranded mRNA Library Prep Kit for NeoPrep and an Illumina NeoPrep Library Prep machine according to manufacturer’s instructions. Phosphate independent small RNA libraries were made as described in (Phillips et al., 2014 (link)) using RNA 5′ polyphosphatase (Illumina) and the Illumina TruSeq Small RNA Library Prep Kit. Libraries were sequenced on the Illumina HiSeq 2000 platform.
Weiser N.E., Yang D.X., Feng S., Kalinava N., Brown K.C., Khanikar J., Freeberg M.A., Snyder M.J., Csankovszki G., Chan R.C., Gu S.G., Montgomery T.A., Jacobsen S.E, & Kim J.K. (2017). MORC-1 integrates nuclear RNAi and transgenerational chromatin architecture to promote germline immortality. Developmental cell, 41(4), 408-423.e7.
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10

RNA Integrity Assessment and RNA-seq

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The quality of RNA was assessed using the Agilent 2100 Bioanalyzer microfluidics-based platform (Agilent Technologies, Inc.). The RNA Integrity Number (RIN) was over 9.0 in all samples. RNA-seq was performed by the DNA Sequencing Section at Okinawa Institute of Science and Technology Graduate University for two biological replicates per condition. One hundred nano grams of total RNA were used for RNA-seq library preparation with a TruSeq Stranded mRNA Library Prep Kit for NeoPrep (NP-202-1001, Illumina), which allows polyA-oligo(dT)-based purification of mRNA, according to the manufacturer’s protocol, with minor modifications and optimization as follows. Custom dual index adaptors were ligated at the 5ʹ and 3ʹ-ends of the library, and PCR was performed for 11 cycles. 150-base-pair pair-end read RNA-seq was performed using a Hiseq 3000/4000 PE Cluster Kit (PE-410-1001; Illumina) and a Hiseq 3000/4000 SBS Kit (300 Cycles) (FC-410-1003; Illumina) on a Hiseq4000 (Illumina), according to the manufacturer’s protocol. Sequence data are available through ArrayExpress under the accession number [E-MTAB-8287].
Mostafa D., Takahashi A., Yanagiya A., Yamaguchi T., Abe T., Kureha T., Kuba K., Kanegae Y., Furuta Y., Yamamoto T, & Suzuki T. (2020). Essential functions of the CNOT7/8 catalytic subunits of the CCR4-NOT complex in mRNA regulation and cell viability. RNA Biology, 17(3), 403-416.
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