Ovation universal rna seq system

Manufactured by Tecan

Sourced in United States, Netherlands

The Ovation Universal RNA-Seq System is a lab equipment product designed for RNA-sequencing applications. It provides a streamlined and efficient workflow for generating high-quality sequencing libraries from a wide range of input RNA samples.

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13 protocols using ovation universal rna seq system

Bacterial rRNA Depletion for Arabidopsis and Lettuce RNA-Seq

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Libraries from Arabidopsis samples were prepared using the Ovation Universal RNA-Seq System (NuGEN, San Carlos, CA, USA), with additional custom AnyDeplete (NuGEN, San Carlos, CA, USA) probes (Nobori et al. 2018 (link)). RNA extracted from leaves inoculated with 1 × 10⁹ CFU/ml were subjected to bacterial rRNA depletion with the Ribo-Zero™ rRNA Removal Kit, Bacteria (Illumina, San Diego, CA, USA). Libraries from lettuce samples were prepared using both Bacteria and Plant Ribo-Zero™ rRNA Removal Kits (Illumina, San Diego, CA, USA). Libraries from bacterial inoculum were prepared using the Ribo-Zero™ rRNA Removal Kits, Bacteria (Illumina, San Diego, CA, USA).
Sequencing libraries were quality controlled and quantified using a combination of Qubit dsDNA HS (Thermo Fisher Scientific, Waltham, MA, USA) and Advanced Analytical Fragment Analyzer High Sensitivity NGS DNA (Agilent, Santa Clara, CA, USA) assays. Sequencing was conducted in an Illumina HiSeq 4000 flow cell with a 1 × 50 -bp single read format using HiSeq 4000 SBS reagents. Base calling was done by Illumina Real Time Analysis (RTA) v2.7.7 and the RTA output was demultiplexed and converted to FastQ format with the Illumina Bcl2fastq v2.19.1 software.

Jacob C., Velásquez A.C., Josh N.A., Settles M., He S.Y, & Melotto M. (2021). Dual transcriptomic analysis reveals metabolic changes associated with differential persistence of human pathogenic bacteria in leaves of Arabidopsis and lettuce. G3: Genes|Genomes|Genetics, 11(12), jkab331.

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Transcriptome Profiling of K562 Cells

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A detailed step-by-step protocol has been deposited in the protocols.io repository (Gressel et al. 2019a ). TT-seq and RNA-seq were performed in 2 biological replicates including RNA spike-ins. Briefly, experiments were performed using

5 \times 10^{7}

K562 cells per biological replicate. Cells were kept at optimal growth conditions and supplemented with 5 µg mL⁻¹ of α-amanitin or solvent (water) for 8 h. After 7 h 55 min, a 4-thiouridine (4sU) labeling pulse (Sigma-Aldrich, T4509) was applied for 5 min using 500 µM (see Supplementary material S3). Total RNA was isolated with the QIAzol reagent (# 79306) according to manufacturer’s instructions except for the addition of 150 ng of RNA spike-in pool with QIAzol reagent as previously described (Schwalb et al. 2016 (link); Gressel et al. 2019a ). The Ovation Universal RNA-Seq System (NuGEN) was used for strand-specific library preparation as described (Gressel et al. 2019b (link)). Purified cDNA libraries were analyzed by Fragment Analyzer prior to Illumina sequencing. Sequencing was performed on a HiSeq 2500 (Illumina) in paired-end mode with 50-bp read length.

Baar T., Dümcke S., Gressel S., Schwalb B., Dilthey A., Cramer P, & Tresch A. (2022). RNA transcription and degradation of Alu retrotransposons depends on sequence features and evolutionary history. G3: Genes|Genomes|Genetics, 12(5), jkac054.

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RNA-seq Library Preparation and Sequencing

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RNA-seq libraries were prepared using the TruSeq standard mRNA library preparation kit [for poly(A)-RNA-seq; Illumina] or the ovation universal RNA-seq system (for rRNA-depleted RNA-seq; NuGEN) and sequenced by the HiSeq 2500 sequencing system (Illumina). Note that except for the results shown in Supplemental Figure S1, D–F, all of our analyses were performed on poly(A)-selected libraries. This choice was made to minimize the number of falsely identified non-XDT genes: Poly(A)-selected libraries contained fewer sporadic intergenic reads than rRNA-depleted libraries in wild-type (XRN2-proficient) conditions. Since readthrough is defined via a comparison between xrn-2-depleted and control conditions, this lower background permitted more facile detection of readthrough (i.e., XDT genes) and thus helped to avoid false-positive identifications of non-XDT genes.

Miki T.S., Carl S.H, & Großhans H. (2017). Two distinct transcription termination modes dictated by promoters. Genes & Development, 31(18), 1870-1879.

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Transient Transcriptome Sequencing Protocol

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TT-seq was performed as described (27 (link),31 (link)), with minor changes. Specifically, 6 × 10⁷ cells, grown in absence of penicillin and streptomycin, from two biological replicates were treated for 15 and 30 min with solvent (DMSO) or 7.5μM 1-NM-PP1 (NM) at 37°C and 5% CO₂. For the 15 min treatment, cells were exposed to 10 min 500 μM of 4-thiouracil (4sU, Carbosynth, NT06186) after 5 min of DMSO or NM treatment (15 min total treatment). For the 30 min treatment, cells were exposed to 10 min 500 μM of 4-thiouracil (4sU, Carbosynth, NT06186) after 20 min of DMSO or NM treatment (30 min total treatment). Cells were lysed in 5 ml of QIAzol (Qiagen) and 300 ng of RNA spike-ins mix were added to each sample. RNA spike-ins were produced as described (27 (link)). RNAs were sonicated to obtain fragments of <10 kb using AFAmicro tubes in a S220 Focused-ultrasonicator (Covaris Inc, parameters: 10 s, peak power 100, cycles 200, duty cycle 1%). 4sU-labeled RNAs were purified from 240 μg of each of the fragmented RNAs. Biotinylation and purification of 4sU-labeled RNAs was performed as described (27 (link),32 (link)). 100 ng of input RNA was used for strand-specific library preparation according to the Ovation Universal RNA-seq System (NuGEN). Libraries were prepared using random hexamer priming only.

Tellier M., Zaborowska J., Caizzi L., Mohammad E., Velychko T., Schwalb B., Ferrer-Vicens I., Blears D., Nojima T., Cramer P, & Murphy S. (2020). CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation. Nucleic Acids Research, 48(14), 7712-7727.

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Prostate Cancer Transcriptome Sequencing

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After prostatectomy, the prostate was fixed with formalin and paraffin. To adequately extract only prostate cancer from formalin-fixed paraffin-embedded tissue, a pathologist identified a site that was morphologically diagnosed with cancer. Library preparation for whole-transcriptome sequencing, with rRNA depletion, was performed using the NuGen Ovation Universal RNA-Seq system (Part#: 0343) according to manufacturer's protocol. The libraries were analyzed on Agilent 4200 TapeStation System using High Sensitivity D1000 ScreenTape Assay (Cat#: 5067-5584) and quantified using KAPA qPCR (Cat# KK4835). Libraries were then normalized to 10nM, and specific number of libraries were combined per pool to get about 100 million reads per sample. Each pooled library was then clustered and sequenced on Illumina NextSeq 550 instrument using NextSeq 500/550 High Output Kit v2 (300 cycles) in 2 × 150 bp paired end sequencing format.

Nagaya N., Rosenfeld J., Lee G.T, & Kim I.Y. (2020). RNA-seq profile of African American men with a clinically localized prostate cancer. Prostate International, 9(3), 125-131.

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Transcriptomic Profiling of Plasmodium-Infected Mosquito Tissues

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We prepared RNA-seq libraries from P. falciparum-infected MGs and SGs obtained in two independent experimental infections (Infections 2 and 3; Supplementary Table S1). Immediately after dissection, tissues were stored in TRIzol (Invitrogen) and frozen at −80°C until subsequent processing. Total RNA was extracted from a pool of ∼30 MGs and a pool of ∼60 SGs from 30 mosquitoes using the TRIzol manufacturer protocol. RNA concentration was quantified using a Qubit^® 2.0 Fluorometer, and RNA integrity was determined with an Agilent 2100 Bioanalyzer. We used the Ovation^® Universal RNA-seq System (Nugen Technologies) for strand-specific RNA-seq library construction following the manufacturer instructions. Custom primers specific to mosquito ribosomal sequences were designed to reduce the percentage of ribosomal reads in the sample and the ribo-depletion step was incorporated into the standard workflow (Supplementary Table S3). Libraries were sequenced at Cabimer (Spain) using an Illumina NextSeq500 for both 2 × 150 bp paired-end and 1 × 75 bp single-end reads.

Ruiz J.L., Ranford-Cartwright L.C, & Gómez-Díaz E. (2021). The regulatory genome of the malaria vector Anopheles gambiae: integrating chromatin accessibility and gene expression. NAR Genomics and Bioinformatics, 3(1), lqaa113.

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Comprehensive RNA-seq Workflow Across Brain Regions

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Total RNA was extracted with TriReagent (Molecular Research Center Inc.) and RNA quality was assessed with a 2100 Bioanalyzer (Agilent Technologies) using Agilent RNA 600 Nano Chip kit (Agilent Technologies). rRNA was depleted with Ribo-Zero Gold rRNA Removal kit (Illumina Inc; mPFC and vHPC) or custom Insert Dependent Adaptor Cleavage (InDA-C) primers (BNST). RNA was fragmented using the S2 ultrasonicator (Covaris Inc.) and sequencing libraries were prepared with Nextera (Illumina; vHPC), ScriptSeq v2 (Epicentre; mPFC), or Ovation Universal RNA-Seq System (NuGEN; BNST) RNA-seq library preparation kits. Libraries were size-selected with Pippin Prep (Sage Science) and sequencing was conducted on HighSeq 2000 (vHPC, paired-end 91 bp, Illumina) or NextSeq 500 platforms (mPFC and BNST, single-end 96 bp; Illumina).
The RNA-seq reads were trimmed for adapters with Cutadapt v1.8.3 (vHPC) and FastX toolkit (mPFC, BNST) and PCR duplicates were removed with PRINSEQ v0.20.4. Reads were aligned using STARv 2.5.0c (Dobin et al., 2013 (link)) with default settings to mouse genome GRCm38, and annotated to gene exons with HTSeq v0.6.1 (Anders et al., 2015 (link)) using GTF release 86 (update 2016-10).

Laine M.A., Trontti K., Misiewicz Z., Sokolowska E., Kulesskaya N., Heikkinen A., Saarnio S., Balcells I., Ameslon P., Greco D., Mattila P., Ellonen P., Paulin L., Auvinen P., Jokitalo E, & Hovatta I. (2018). Genetic Control of Myelin Plasticity after Chronic Psychosocial Stress. eNeuro, 5(4), ENEURO.0166-18.2018.

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Transcriptome Profiling of Meloidogyne incognita

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Total RNAs were extracted from 4 M. incognita developmental stages (pre-parasitic J2s, parasitic J3-J4, adult females and eggs) using TRIzol Reagent (Invitrogen); three independent biological replicates were performed for each stage. Total RNA quality and quantity were assessed by a 2100 Bioanalyser (Agilent technologies). Samples with RNA integrity number (RIN) over 8.5 were kept for cDNA library construction, except for eggs samples for which RIN ranged between 6 and 7. An input of 100 ng total RNA was provided to construct cDNA libraries via the Ovation Universal RNAseq system (Nugen technologies). To eliminate unwanted rRNA transcripts, we designed 101 InDA-C primers to target M. incognita 28S and 18S transcripts for depletion. The 12 cDNA libraries (4 stages x 3 replicates) were quantified and equilibrated to 4 nM using Kapa QPCR (Kapa Biosystems). Finally, multiplexed libraries were sequenced on an Illumina NextSeq 500 sequencer on two High 150 flow cells (400M PE75 reads), on the UCA Genomix sequencing platform of Nice Sophia-Antipolis.

Blanc-Mathieu R., Perfus-Barbeoch L., Aury J.M., Da Rocha M., Gouzy J., Sallet E., Martin-Jimenez C., Bailly-Bechet M., Castagnone-Sereno P., Flot J.F., Kozlowski D.K., Cazareth J., Couloux A., Da Silva C., Guy J., Kim-Jo Y.J., Rancurel C., Schiex T., Abad P., Wincker P, & Danchin E.G. (2017). Hybridization and polyploidy enable genomic plasticity without sex in the most devastating plant-parasitic nematodes. PLoS Genetics, 13(6), e1006777.

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RNA-Seq of Dendritic Cell Subsets

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Total RNA was extracted from CD1c⁺DCs IA SF, IA peripheral blood, HC peripheral blood, and CD141⁺ DCs IA SF. RNA samples were reverse transcribed and sequencing libraries constructed using NuGen Ovation Universal RNA-Seq System (CAT# 0402-A01) according to the manufacturer’s protocol. The resulting sequencing libraries were analyzed using the Caliper LabChip GX and quantified using KAPA qPCR. Libraries were then normalized and pooled in one batch of six and one batch of five. Each pool was clustered and sequenced on an Illumina NextSeq500 instrument using 2×100 bp paired-end reads, following the manufacturer’s protocols. The average number of reads per sample was 117 million reads with a minimum of 90.1 million reads. Raw read quality was evaluated using FastQC, and statistical analysis was performed as previously described (6 (link), 18 (link)). Data are available on NCBI GEO with the accession number GSE151897 and GSE108174.

Canavan M., Marzaioli V., Bhargava V., Nagpal S., Gallagher P., Hurson C., Mullan R., Veale D.J, & Fearon U. (2021). Functionally Mature CD1c+ Dendritic Cells Preferentially Accumulate in the Inflammatory Arthritis Synovium. Frontiers in Immunology, 12, 745226.

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4tU-seq for Transcription Dynamics

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The 4tU-seq experiments and analyses were performed as previously described (Eser et al., 2016 (link); Miller et al., 2011 (link)). The Spt5 depletion strain was grown as described earlier and cultures were labeled with 4tU for 10 minutes at 0 and 4.5 hours after the addition of thiamine and auxin. RNA spike-ins were added to cell pellets at the first step of RNA purification (Schwalb et al., 2016 (link)). The amount of spike-ins was adjusted to the cell number for each sample (120 ng of spike-in mix for 2.5 × 10^8 cells for all samples). Sequencing libraries were prepared according to the manufacturer’s recommendations using the Ovation Universal RNA-Seq System (NuGen). Libraries were sequenced on an Illumina HiSeq 2500 at LAFUGA, Ludwig-Maximilians-University of Munich.

Shetty A., Kallgren S.P., Demel C., Maier K.C., Spatt D., Alver B.H., Cramer P., Park P.J, & Winston F. (2017). Spt5 plays vital roles in the control of sense and antisense transcription elongation. Molecular cell, 66(1), 77-88.e5.

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