Ion onetouch 200 template kit v2

For the clonal amplified sequencing templates, the libraries were pooled in equimolar amounts and emulsion PCR on Ion OneTouch system with Ion OneTouch 200 Template kit v2 (Life Technologies, Gaithersburg, MD). The templates were automatically enriched with the Ion OneTouch ES system (Life Technologies). Next Generation Sequencing was performed using the Ion Torrent Personal Genome Machine (PGM) sequencer system using a 316D sequencing chip (Life Technologies)⁶⁷ . Raw sequence data were analyzed by Ion Torrent Suite v4.0.2.

Total RNA was isolated employing Ultrasense Viral RNA kit (Qiagen, Germany) and concentrated using Ribominus Concentration module (Life technologies, USA). Ribosomal RNA was depleted using Ribominus Eukaryotic kit v2 (Life technologies, USA). Purified mRNA was fragmented using NEBNext^® Magnesium RNA Fragmentation Module (New England Biolabs, USA). After adaptor ligation of a library of 200 bp size, cDNA was synthesized and amplified using Ion total RNA sequencing kit v2 (Life technologies, USA) as per manufacturer’s instructions. The cDNA library was quantified and size distribution analysed on High sensitivity DNA chip kit on Agilent Bioanalyzer 2100. Emulsion PCR was carried out using the Ion OneTouch™ 200 Template Kit v2 (Life Technologies, USA) according to the manufacturer’s instructions. Sequencing of the cDNA libraries were carried out on 316/318 chips using the Ion Torrent PGM system and employing the Ion Sequencing 200 kit (Life Technologies, USA) according to the supplier’s instructions. After sequencing, low quality reads and polyclonal sequences were filtered by the PGM software. Sequences matching the PGM 3′ adaptor were also automatically trimmed.

The messenger RNA from chick skeletal muscle cells was extracted and isolated by Dynabeads mRNA DIRECT Micro Kit (Invitrogen). Samples were pooled from three biological replicates each. The library preparation was carried out using the Ion Total RNA-Seq Kit v2 (Life Technologies) with 500 ng of PolyA (RNA) according to manufacturer’s instructions. To assess the yield and size distribution of the fragmented RNA we used Qubit RNA Assay Kit (Invitrogen) and Agilent RNA 6000 Pico Kit (Agilent, GE). The complementary DNA (cDNA) was amplified without barcoding. The dsDNA HS Assay Kit (Invitrogen) and Agilent High Sensitivity DNA Kit (Agilent, GE) were used to assess the yield and size distribution of the amplified DNA. The template was made in One Touch System by Ion One Touch 200 template kit V2 (Life technologies) according to manufacturer’s instructions. The transcriptome sequencing was performed using Ion PGM 200 Sequencing Kit (Life Technologies) with 318 chips in Ion Torrent PGM machine.

Five patients were randomly selected from each groups to examine the expression of their exosomal miR. The volumes of the RNA samples (collected from 250-μl serum samples) was normalized. RNA libraries were generated using an Ion Total RNA-Seq Kit v2 (Life Technologies) in accordance with the manufacturer’s instructions. The RNA libraries were then processed for the emulsion PCR using an Ion OneTouchTM system and an Ion OneTouch 200 Template kit v2 (Life Technologies). Template-positive Ion SphereTM particles were enriched and purified for the sequencing reaction with an Ion OneTouchTM ES system (Life Technologies). The template-positive Ion SphereTM Particles were then applied to Ion PI™ Chips (Life Technologies), and the next-generation sequencing reaction was carried out using an Ion Proton™ Semiconductor sequencer (Life Technologies). All of the sequencing data were mapped on a miR sequence using the CLC Genomics Workbench software program (CLC Bio, Aarhus, Denmark), and an expression analysis was performed for each sample.

Fragment DNA library construction was performed using the Ion Xpress Plus Fragment Library Kit (Life Technologies). 100 ng of genomic DNA was used for the enzymatic shearing and ligation of Ion Torrent™ adapters according to the manufacturer's instructions. Fragments of 330 bp were selected by 2% agarose gel electrophoresis using the E-Gel iBase™ Power System and E-Gel Safe Imager™ (Invitrogen, Life Technologies). The quality and quantity of the genomic DNA library was assessed by analysing them in High Sensitivity DNA chip in the 2100 Bioanalyzer (Agilent). Template preparation for sequencing was made in The Ion OneTouch™ 2 System by the Ion OneTouch 200 Template Kit v2 (Life Technologies) according to manufacturer's instructions.
Sequencing was performed on the Ion Torrent Personal Genome Machine (PGM) platform using the Ion PGM 200 Sequencing Kit and an Ion 318 chip (Life Technologies).