The library for DNA sequencing using NGS was generated using the Fluidigm Access Array microfluidic chip as previously described38 (link). The confirmed target-specific ACE2 primers were then tagged with Fluidigm specific tag sequences and used for targeted next-generation sequencing38 (link). The prepared amplicon libraries were purified using AMPure XP beads (Beckman Coulter, USA) and quantified using a High Sensitivity DNA assay kit on BioAnalyzer (Agilent, USA). The libraries were further diluted to 1 pg for direct input into the emulsion polymerase chain reaction (PCR) with Ion SphereTM particles using the Ion Template OT2 kit (Ion OneTouch™ instrument). Enrichment of the clonal beads was carried out using the Ion OneTouch™ ES system following the manufacturer’s instructions (Thermo Fischer, USA). The pooled libraries were then sequenced using the Ion 520™ Chip on the Ion S5 XL Semiconductor sequencer, following the manufacturer’s instructions (Thermo Fisher).
Ion onetouch es system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ion OneTouch ES system is a laboratory instrument designed for the automated preparation of Ion Torrent sequencing libraries. The system performs essential steps in the library preparation workflow, including template preparation and enrichment. It is a self-contained, benchtop instrument that streamlines the library preparation process to improve efficiency and consistency.
Automatically generated - may contain errors
Lab products found in correlation
Ion onetouch 2 system, by Thermo Fisher Scientific (4 mentions)
Ion 520 chip, by Thermo Fisher Scientific (2 mentions)
Ion onetouch 2, by Thermo Fisher Scientific (2 mentions)
Ion pgm sequencer, by Thermo Fisher Scientific (2 mentions)
Ion proton semiconductor sequencer, by Thermo Fisher Scientific (2 mentions)
Ion pi chip, by Thermo Fisher Scientific (2 mentions)
Ion onetouch 200 template kit v3, by Thermo Fisher Scientific (2 mentions)
Ion total rna seq kit v2, by Thermo Fisher Scientific (2 mentions)
Ion one touch, by Thermo Fisher Scientific (2 mentions)
Ion pi template ot2 200 kit v3, by Thermo Fisher Scientific (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Dynabeads myone streptavidin c1 beads, by Thermo Fisher Scientific (2 mentions)
Ion onetouch system, by Thermo Fisher Scientific (2 mentions)
Ion 318 chip, by Thermo Fisher Scientific (2 mentions)
Ion pgm 200 sequencing kit, by Thermo Fisher Scientific (2 mentions)
Ion onetouch 200 template kit v2, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Ion s5 xl semiconductor sequencer, by Thermo Fisher Scientific (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
16 protocols using ion onetouch es system
1
NGS-based ACE2 Amplicon Sequencing
2
Bacterial DNA Extraction and 16S rRNA Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BALF pellets were obtained, and bacterial DNA was extracted via centrifugation at 6000 rpm for 5 min at 4 °C, using NucleoSpin Microbial DNA (Macherey-Nagel, Düren, Germany). Extracted DNA was subjected to PCR to amplify the V5–V6 region of the 16S rRNA gene using specific primers via next-generation sequencing (NGS) with the 784F and NGS.1061R primers, including the adaptor and barcode sequences for NGS (Additional file 1 : Table S1). The PCR products were size-selected and purified. Sample libraries were prepared, and the NGS was performed per the method proposed by Akiyama K et al. [17 (link)], using the Ion One Touch, the Ion OneTouch ES system, and the Ion Torrent Personal Genome Machine (Thermo Fisher Scientific, Waltham, MA, USA).
3
Targeted Sequencing with Ion AmpliSeq
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Targeted sequencing was performed as previously described [25 (link)]. Multiplex polymerase chain reaction (PCR) of these panels was performed using the Ion AmpliSeq Library Kit 2.0 (Thermo Fisher Scientific). Primer sequences were digested with FuPa reagent (Thermo Fisher Scientific), and then barcoded using Ion Xpress Barcode Adapters (Thermo Fisher Scientific). Purification was carried out by Agencourt AMPure XP reagents (Beckman Coulter, Brea, CA). The library concentration was determined using an Ion Library Quantitation Kit (Thermo Fisher Scientific); each library was diluted to 10 pM, and the same amount of libraries was pooled for one sequence reaction. Emulsion PCR was carried out using the Ion OneTouch System and Ion PGM Template OT2 200 kit or Ion PI Template OT2 200 Kit v3 (Thermo Fisher Scientific). Template-positive Ion Sphere Particles were then enriched using the Ion OneTouch ES system (Thermo Fisher Scientific), and purified Ion Sphere particles were loaded on an Ion 318 Chip v2 or PI Chip (Thermo Fisher Scientific). Massively parallel sequencing was carried out on Ion PGM or Ion Proton systems (Thermo Fisher Scientific).
4
Targeted Genomic DNA Profiling from Blood
5
Targeted Sequencing of Neuromuscular Disorders
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 123 cases (includes 102 DMD/BMD/Carrier, 2 UMD, and 19 LGMD) were screened by targeted sequencing using a custom amplicon panel. For each sample, 50 ng of DNA was amplified with custom primer pools using Ion Ampliseq™ HiFi Mix (Thermo Fisher Scientific, IN). This was followed by partial digestion, adaptor + barcode ligation, and library amplification. Libraries were purified using AgencourtAMPure XP (Beckman Coulter, United States). Purified libraries were quantified by Qubit dsDNA HS assay kit (Thermo Fisher Scientific, IN) and then pooled in equimolar concentrations. Emulsion PCR of pooled and diluted libraries was carried out using the Ion OneTouch™ 2 System (Thermo Fisher Scientific, IN) followed by enrichment of template-positive Ion Sphere™ Particles on an Ion OneTouch™ ES system (Thermo Fisher Scientific, IN). Sequencing was carried out on the Ion Proton™ and Ion S5™ systems using Ion PI and Ion 530 chips respectively, with an average depth of 80x.
6
NGS library preparation and sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified DNA (50 ng) was used to construct DNA fragment libraries using the Ion Xpress Plus Fragment Library Kit, following the manufacturer’s protocol (Thermo Fisher Scientific). An Ion PGM Hi-Q OT2 Kit was used to prepare template-positive Ion Sphere Particles. Thermal cycling amplification of DNA fragments in microreactors was performed using the Ion OneTouch 2 system (Thermo Fisher Scientific). Positive particles were isolated and purified employing the Ion OneTouch ES system (Thermo Fisher Scientific). The particles were loaded onto Ion 318 chips and sequenced using an Ion PGM Sequencer (Thermo Fisher Scientific). Sequencing of the libraries was separately performed in three independent chips together with other samples for different research projects.
7
Ion Torrent PGM Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The emulsion PCR was carried out with the Ion OneTouch 2 System and Ion Torrent PGM Hi-Q OT2 Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s procedure. Template-positive Ion Sphere™ Particles were enriched with the Dynabeads MyOne Streptavidin C1 Beads (Thermo Fisher Scientific, Waltham, MA, USA) and washed with Ion OneTouch Wash Solution. This process was performed using an Ion OneTouch ES system (Thermo Fisher Scientific, Waltham, MA, USA). After the Ion Sphere Particle preparation, Massive Parallel Sequencing was carried out with Ion Torrent PGM sequencer system (Thermo Fisher Scientific, Waltham, MA, USA) using the Ion Torrent PGM Hi-Q Sequencing Kit and Ion 314v2 or 316v2 Chip (Thermo Fisher Scientific, Waltham, MA, USA), according to the established procedures.
8
Targeted Sequencing of ACE2 and TMPRSS2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primer sets were designed to cover the exonic and intronic regions of the ACE2 and TMPRSS2 genes. The details of the primers are listed in Supplementary Table S1. The targeted amplicon size was about 200 bp. First, all primers were assessed using control DNA samples, and thus, the expected amplicon sizes were validated using conventional PCR. Then, the library for targeted nextgeneration sequencing was prepared using the Fluidigm 48.48 Access Array integrated fluidic circuit (IFC) (Fluidigm Europe B.V, Netherlands) as previously described (10) (link). The purified amplicon libraries were diluted to 1 pg and then amplified using emulsion PCR with Ion Template OT2 kit in Ion OneTouch ™ ES system following the manufacturer's instructions (ThermoFisher, USA). Next, all samples of COVID-19 patients and healthy controls were equally pooled and sequenced using the Ion 520 ™ Chip on an Ion S5 XL Semiconductor (ThermoFisher, USA).
9
RNA-Seq Protocol for Differential Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Basically, the analysis was performed using methods previously reported by our group23 (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.
10
RNA-seq Analysis of SUIT-2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!