Whole transcriptome RNA-seq of PAO1 planktonic and biofilm cells was carried out using the ION Torrent Personal Genome System (Life Technologies)90 (link). Following depletion of rRNA using RiboZero (illumina), the cDNA libraries were constructed using the Ion Total RNA-Seq kit v2 (Life technologies) according to the manufacturer’s protocol. The cDNA libraries were used to prepare sequencing templates using the Ion PGM Template OT2 200 kit on the Ion OneTouch™2 instrument (Life Technologies), followed by Ion Sphere™ Quality Control Kit assessment and template-positive Ion Sphere™ Particle (ISP) enrichment using the Ion OneTouch™ ES instrument. Sequencing was carried out using the Ion Sequencing 200 Kit v2 and the Ion 316v2 Chip on the Ion Torrent PGM sequencing platform (Life Technologies). RNA-seq was performed in triplicate using biological replicates. The raw sequencing data are available in the NCBI short read archive (http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi ) with accession number SRP096901 under BioProject PRJNA362216.
Ion sequencing 200 kit v2
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ion Sequencing 200 Kit v2 is a laboratory equipment product designed for ion sequencing applications. It provides the essential components required to perform ion-based DNA sequencing. The kit includes reagents, consumables, and protocols to enable researchers to conduct sequencing experiments. The core function of this product is to facilitate ion-based DNA sequencing workflows in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Ion onetouch 2 instrument, by Thermo Fisher Scientific (2 mentions)
Ion onetouch es instrument, by Thermo Fisher Scientific (2 mentions)
Ion pgm template ot2 200 kit, by Thermo Fisher Scientific (2 mentions)
Ion torrent pgm sequencing platform, by Thermo Fisher Scientific (2 mentions)
Ion total rna seq kit v2, by Thermo Fisher Scientific (2 mentions)
Ion sphere particle, by Thermo Fisher Scientific (2 mentions)
Ion torrent personal genome system, by Thermo Fisher Scientific (2 mentions)
Ion sphere quality control kit assessment, by Thermo Fisher Scientific (2 mentions)
Ribo zero, by Illumina (1 mentions)
Ion onetouch 200 template kit v2 dl, by Thermo Fisher Scientific (1 mentions)
Ion 318 chip, by Thermo Fisher Scientific (1 mentions)
4 protocols using ion sequencing 200 kit v2
1
Whole Transcriptome RNA-seq of Planktonic and Biofilm PAO1
2
Ion Torrent Sequencing Protocol
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Emulsion PCR was performed using the Ion OneTouch 200 Template Kit v2 DL (catalog No. MAN0006957; Life Technologies, USA). Sequencing of the amplicon libraries was performed on a 314 chip with the Ion Torrent personal genome machine system using the Ion Sequencing 200 kit v2 (catalog No. MAN0007273; Life Technologies, USA).
3
Ion Torrent Transcriptome Sequencing Protocol
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Whole transcriptome RNA-Seq was carried out using the ION Torrent Personal Genome System (Life Technologies). Following rRNA depletion and BioAnalyzer quality assessment performed as discussed above, the cDNA libraries was constructed using the Ion Total RNA-Seq kit v2 (Life technologies) according to the manufacturer’s protocol. Following BioAnalyzer size distribution assessment and Qubit quantitation, the cDNA libraries were used to prepare sequencing templates using the Ion PGM Template OT2 200 kit on the Ion OneTouch™2 instrument (Life Technologies), followed by Ion Sphere™ Quality Control Kit assessment and template-positive Ion Sphere™ Particle (ISP) enrichment using the Ion OneTouch™ ES instrument. Sequencing was carried out using Ion Sequencing 200 Kit v2 and the Ion 318 Chip on the Ion Torrent PGM sequencing platform (Life Technologies). For each rRNA depletion method, RNA-sequencing was performed in duplicate using distinct biological replicates. The raw sequencing data are available in the NCBI short read archive (http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi ) with accession number SRP072237 under BioProject PRJNA316121.
4
Ion Torrent PGM Sequencing Protocol
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The adapted barcoded libraries were equalized using the Ion Library Equalizer kit to a final concentration of 100 pM, pooled and diluted to 26 pM, and attached to the surface of Ion Sphere particles (ISPs) using an Ion Personal Genome Machine (PGM) Template OT2 200bp kit v2 (Life Technologies) according to the manufacturer’s instructions, via emulsion PCR. Quality of ISPs templates was checked using Ion Sphere Quality Control Kit (part no. 4468656) with the Qubit 2.0 device. Sequencing of the pooled libraries was carried out on the Ion Torrent PGM system using the Ion Sequencing 200 kit v2 (all from Life Technologies) for 150 cycles (600 flows), with a 318-chip following the manufacturer’s instructions. Demultiplexing and classification were performed using the QIIME v.1.1.1.1 platform [6] (link). The resulting sequence data were trimmed to remove adapters, barcodes, and primers during the demultiplexing process. In addition, bioinformatic process filters were applied to the sequence data for the removal of low-quality reads below Q25 Phred score and denoised to exclude sequences with read length below 100 bp. De novo operation taxonomic units (OTUs) were clustered using the Uclust algorithm and defined by 97% sequence similarity.
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