Ion onetouch 200 template kit v3

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Ion OneTouch 200 Template kit v3 is a laboratory equipment product designed for the preparation of DNA templates for sequencing on Ion Torrent platforms. The kit provides the necessary reagents and consumables to perform template preparation, enrichment, and recovery steps required for Ion Torrent sequencing workflows.

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Ion OneTouch 200 Template kit (9 citations) OneTouch Ion™ Template Kit (4 citations)

5 protocols using ion onetouch 200 template kit v3

Ion Proton Sequencing of RNA Libraries

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RNA libraries were generated using an Ion Total RNA-Seq Kit v2 (ThermoFisher Scientific) according to the manufacturer’s instructions. The RNA libraries were then processed for an emulsion polymerase chain reaction (PCR) using an Ion OneTouch^TM system and an Ion OneTouch 200 Template kit v3 (ThermoFisher Scientific). Template-positive Ion Sphere^TM particles were enriched and purified for the sequencing reaction with an Ion OneTouch^TM ES system (ThermoFisher Scientific). The template-positive Ion Sphere^TM Particles were then applied to Ion PI^TM Chips (ThermoFisher Scientific), and high-throughput sequencing was performed using an Ion Proton™ Semiconductor sequencer (ThermoFisher Scientific). All of the sequencing data were mapped on a human reference genome sequence (GRCh37/hg19) using the Torrent Suite software program (ThermoFisher Scientific). An expression analysis of each sample was imported into the CLC Genomics Workbench software program (CLC bio, Aarhus, Denmark), and the significance of the differences among the samples was determined by an unpaired t-test.

Konishi H., Fujiya M., Kashima S., Sakatani A., Dokoshi T., Ando K., Ueno N., Iwama T., Moriichi K., Tanaka H, & Okumura T. (2020). A tumor-specific modulation of heterogeneous ribonucleoprotein A0 promotes excessive mitosis and growth in colorectal cancer cells. Cell Death & Disease, 11(4), 245.

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RNA-Seq Protocol for Differential Gene Expression

Cited 1 time

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Basically, the analysis was performed using methods previously reported by our group^{23 (link)}. RNA libraries were generated using an Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Emulsion PCR was carried out with an Ion OneTouchTM system and an Ion OneTouch 200 Template Kit v3 (Thermo Fisher Scientific). Template-positive Ion Sphere™ particles were enriched and purified for the sequencing reaction with an Ion OneTouch™ ES system (Thermo Fisher Scientific). The template-positive Ion Sphere™ Particles were loaded onto Ion PI™ Chips (Thermo Fisher Scientific) and for high throughput sequencing with an Ion Proton™ Semiconductor sequencer (Thermo Fisher Scientific). Sequencing data were mapped on a human reference genome sequence (GRCh38/hg38) using the Torrent Suite software program (Life Technologies). The expression analysis was performed in the CLC Genomics Workbench software program (CLC bio, Aarhus, Denmark), and differences among the samples were determined using an unpaired Student’s t-test. The gene list describing fold change and p-value was uploaded to the MetaCore software (Clarivate Analytics, PA, USA, URL; https://portal.genego.com/, version 6.33.69110.), and then pathway analysis was performed.

Kojima K., Konishi H., Momosaki K., Komatani Y., Katsuyama A., Nakagawa K., Kanamitsu K., Yakushiji F., Fujiya M, & Ichikawa S. (2024). Synthesis and biological evaluation of echinomycin analogues as potential colon cancer agent. Scientific Reports, 14, 7628.

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RNA-seq Analysis of SUIT-2 Cells

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Total RNA from SUIT-2 cells was extracted using an RNeasy mini kit (Qiagen, Inc.) according to the manufacturer's protocols. RNA libraries were generated using an Ion Total RNA-Seq kit v2 (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The RNA libraries were then processed for emulsion PCR using an Ion OneTouch™ system and an Ion OneTouch 200 Template kit v3 (Thermo Fisher Scientific, Inc.). Template-positive Ion Sphere™ particles were enriched and purified for the sequencing reaction with an Ion OneTouch ES system (Thermo Fisher Scientific, Inc.). The template-positive Ion Sphere Particles were then applied on Ion PI™ Chips (Thermo Fisher Scientific, Inc.), and a high throughput sequencing reaction was performed using an Ion Proton™ Semiconductor sequencer (Thermo Fisher Scientific, Inc.). All of the sequencing data were mapped on a human reference genome sequence (GRCh38/hg38), the expression analysis and gene functional annotation analysis for each sample was imported into CLC Genomics Workbench software v9.0.1 (CLC bio; Qiagen Digital Insights), and significant differences between the samples were determined using unpaired Student's t-tests.

Kita A., Fujiya M., Konishi H., Tanaka H., Kashima S., Iwama T., Ijiri M., Murakami Y., Takauji S., Goto T., Sakatani A., Ando K., Ueno N., Ogawa N, & Okumura T. (2020). Probiotic-derived ferrichrome inhibits the growth of refractory pancreatic cancer cells. International Journal of Oncology, 57(3), 721-732.

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High-throughput Custom Amplicon Sequencing

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Using 50 ng of each DNA sample, an ultra-high multiplex polymerase chain reaction (PCR) was performed and a DNA fragments library (5 primer pools per sample) was generated, using an Ion AmpliSeqTM Library Kit 2.0 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions in order to perform custom amplicon sequencing. The concentration and quality of the DNA fragments library was evaluated with an Agilent 2200 Tape station (Agilent Technologies, Santa Clara, CA, USA). The DNA fragment libraries were then processed for an emulsion PCR using an Ion OneTouchTM System and an Ion OneTouch 200 Template Kit v3 (Life Technologies, Carlsbad, CA, USA). Template-positive Ion SphereTM Particles from the sequencing reaction were enriched and purified with an Ion OneTouchTM ES system (Life Technologies, Carlsbad, CA, USA). The template-positive Ion SphereTM Particles were then applied on Ion PI™ Chips (Life Technologies, Carlsbad, CA, USA), and the high throughput sequencing reaction was carried out using an Ion Proton™ Semiconductor sequencer (Life Technologies, Carlsbad, CA, USA).

Fujibayashi S., Sasajima J., Goto T., Tanaka H., Kawabata H., Fujii T., Nakamura K., Chiba A., Yanagawa N., Moriichi K., Fujiya M, & Kohgo Y. (2016). A high-throughput sequence analysis of Japanese patients revealed 11 candidate genes associated with type 1 autoimmune pancreatitis susceptibility. Biochemistry and Biophysics Reports, 6, 76-81.

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RNA Sequencing for Transcriptome Analysis

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RNA was purified using a mirVana miRNA isolation kit (Thermo Fisher Scientific). RNA libraries were generated using an Ion Total RNA-Seq kit v2 (Life Technologies) according to the manufacturer’s instructions. The RNA libraries were then processed for emulsion PCR using an Ion OneTouch^TM system and an Ion OneTouch 200 Template kit v3 (Life Technologies). Template-positive Ion Sphere^TM particles were enriched and purified for the sequencing reaction with an Ion OneTouch^TM ES system (Life Technologies). The template-positive Ion Sphere^TM particles were then applied on Ion PI™ chips (Life Technologies) and a high-throughput sequencing reaction was carried out using an Ion Proton™ semiconductor sequencer (Life Technologies). All of the sequencing data were mapped on a human reference genome sequence (GRCh37/hg19) using the Torrent Suite software program (Life Technologies). The expression analysis for each sample was imported into the CLC Genomics Workbench software program (CLC Bio, Aarhus, Denmark) and the significance of the differences among the samples was determined by an unpaired t-test. The gene ontology (GO) analysis was performed using the MetaCore software.

Takahashi K., Fujiya M., Konishi H., Murakami Y., Iwama T., Sasaki T., Kunogi T., Sakatani A., Ando K., Ueno N., Kashima S., Moriichi K., Tanabe H, & Okumura T. (2020). Heterogenous Nuclear Ribonucleoprotein H1 Promotes Colorectal Cancer Progression through the Stabilization of mRNA of Sphingosine-1-Phosphate Lyase 1. International Journal of Molecular Sciences, 21(12), 4514.

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