Ovation sp ultralow library system

Manufactured by Tecan

The Ovation SP Ultralow Library System is a lab equipment product designed for library preparation workflows. It provides an automated solution for generating high-quality libraries from low-input samples. The system is capable of processing a wide range of input materials, including genomic DNA, RNA, and FFPE samples.

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Lab products found in correlation

Kapa stranded mrna seq kit, by Roche (2 mentions) Kapa mrna capture beads, by Roche (2 mentions) Ovation rna seq system v2, by Tecan (2 mentions) Rna6000 picoassay for the bioanalyzer 2100, by Agilent Technologies (2 mentions) Mondrian sp workstation, by Tecan (2 mentions) Pyromark q96 id, by Qiagen (1 mentions) Epitect bisulfite kit, by Qiagen (1 mentions) Pyromark pcr kit, by Qiagen (1 mentions) Kapa hifi uracil, by Roche (1 mentions) Pyromark assay design software, by Qiagen (1 mentions) Hiseq 2000 or 2500, by Illumina (1 mentions) Agencourt ampure xp bead, by Beckman Coulter (1 mentions) Quant it picogreen dsdna assay, by Thermo Fisher Scientific (1 mentions) Casava, by Illumina (1 mentions) Nebnext poly a mrna magnetic isolation module, by New England Biolabs (1 mentions)

4 protocols using ovation sp ultralow library system

moDC and PBMC RNA Sequencing

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For moDC analysis, RNA quality was ensured using the RNA6000 PicoAssay for the Bioanalyzer 2100 (Agilent), followed by RNA amplification using the Ovation RNA-Seq System V2 (NuGen Technologies, San Carlos, CA) and library preparation using the Ovation SP Ultralow Library System (NuGen) according the manufacturers’ protocols. For PBMC analysis, mRNA was enriched using KAPA mRNA capture beads followed by preparation of libraries using the KAPA Stranded mRNA Seq kit (Kapa Biosystems, Wilmington, MA) according the manufacturers’ protocols.

Veenbergen S., Li P., Raatgeep H., Lindenbergh-Kortleve D., Simons-Oosterhuis Y., Farrel A., Costes L., Joosse M., van Berkel L., de Ruiter L., van Leeuwen M., Winter D., Holland S., Freeman A., Wakabayashi Y., Zhu J., de Ridder L., Driessen G., Escher J., Leonard W, & Samsom J. (2019). IL-10 signaling in dendritic cells controls IL-1β-mediated IFNγ secretion by human CD4+ T cells: relevance to inflammatory bowel disease. Mucosal immunology, 12(5), 1201-1211.

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moDC and PBMC RNA Sequencing

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Genome-wide DNA Methylation Analysis

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One-hundred nanograms of DNA was bisulfite-converted using the Epitect bisulfite kit (Qiagen). Pyrosequencing assays were designed using Pyromark assay design software (Qiagen). For pyrosequencing, template DNA was amplified using the Pyromark PCR kit (Qiagen), and sequencing was performed using the PyroMark Q96 ID (Qiagen). For bisulfite sequencing, template DNA was amplified using KAPA HIFI Uracil⁺ (KAPA), sequencing libraries were made using the Ovation SP ultralow library system and Mondrian SP⁺ Workstation (NuGEN), and 150-bp paired-end sequencing was performed on the MiSeq (Illumina). Converted sequences were aligned to the mouse genome (mm9) using BS Seeker (Chen et al. 2010 (link)). Primer sets can be found in the Supplemental Material (Supplemental Table 6).

Sheaffer K.L., Kim R., Aoki R., Elliott E.N., Schug J., Burger L., Schübeler D, & Kaestner K.H. (2014). DNA methylation is required for the control of stem cell differentiation in the small intestine. Genes & Development, 28(6), 652-664.

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RNA-seq library preparation and sequencing

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mRNA was purified from 1μg of total RNA extracted as above using a NEBNext Poly(A) mRNA Magnetic isolation module (New England Biolabs). First and second strand cDNA was synthesized using NEBNext synthesis modules (New England Biolabs), purified using Agencourt AMPure XP beads (Beckman Coulter), and quantified using Quant-iT PicoGreen dsDNA assay (Life Technologies). Libraries were prepared from 10ng ds-cDNA using an Ovation SP ultralow library system (Nugen) in combination with a Mondrian SP work station (Nugen). Libraries were amplified by PCR for 15 cycles, quantified, denatured and 10pmol of each library were pooled and sequenced on a 1x50bp program on a Hiseq 2000 or 2500 (Illumina). After sequencing, the BCL file was demulitiplexed and converted to a FASTQ file using CASAVA (Illumina). Files were aligned to the mm10 UCSC genome using TopHat splice junction mapper with Bowtie2 [21 (link)]. Features were counted and a count table produced using Rsubread[22 (link)]. Raw and processed data including batch information and output from differential expression analysis with DESeq2 is available in the GEO repository (https://www.ncbi.nlm.nih.gov/geo) under series record GSE98492.

Ferdinand J.R., Richard A.C., Meylan F., Al-Shamkhani A, & Siegel R.M. (2018). Cleavage of TL1A differentially regulates its effects on innate and adaptive immune cells. Journal of immunology (Baltimore, Md. : 1950), 200(4), 1360-1369.

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