Ion onetouch 200 template kit v2 dl

The final library was purified using a highly sensitive 2% agarose gel stained with SYBR GOLD DNA (E-Gel™ EX, 2%, Invitrogen™, Cat. G401002, Waltham, MA, USA). The DNA library concentration and final size fragment were measured with 2100 Bioanalyzer Instrument (Agilent Technologies, Santa Clara, CA, USA) fragment analyzer, the resulting average size of the library was 263 bp. PCR emulsion was carried out using Ion OneTouchTM 200 Template Kit v2 DL (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. Enrichment of the amplicon with ionic spheres was carried out using Ion OneTouch ES (Life Technologies, Carlsbad, CA, USA). Sequencing was performed using the Ion 318 Kit V2 Chip (Cat. 4488146, Life Technologies, Carlsbad, CA, USA) and the Ion Torrent PGM system v4.0.2. After sequencing, the readings were filtered by the PGM software to remove the polyclonal (homopolymers > 6) and low-quality sequences (quality score ≤ 20).

Total RNA was isolated with Trizol in accordance with the manufacturer’s protocol (Life Technologies, Carlsbad, CA, USA) and treated with DNase I (Epicentre, Madison, WI, USA) to remove genomic DNA contamination. RNA purity was quantified using Qubit (Life Technologies). Seven micrograms of total RNA were used for ribosomal RNA (rRNA) depletion with MICROBExpress^TM kit (Thermo Fisher Scientific Inc., Idstein, Germany). The efficiency of depletion was evaluated in agarose gel electrophoresis (1%), followed by quantification of the total RNA with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). A total of 500 ng mRNA was used for the construction of a sequencing library using the standard protocol of the SOLid Total RNAseq Kit (Life Technologies). The libraries were barcoded using the SOLiD Transcriptome Multiplexing Kit (Life Technologies). The emulsion PCR and sequencing were performed according to the Ion One Touch^TM 200 Template Kit v2 DL and the Ion PI^TM Sequencing 200 Kit v2 using the standard Life Technologies protocols, respectively. The Ion Proton Semiconductor Sequence (Life Technologies) was used to sequence 12 libraries generated from three biological replicates from each independent treatment.

The genome library preparation was performed by using the Ion Xpress^TM Plus Fragment Library Kit (Life Technologies, USA) following the manufacturer’s protocol, 4471989 Rev E. The sheared DNA was separated using a 2% agarose gel (Promega, USA) and fragments that corresponded to 200 bp of a sequencing run (approximately 330 bp) were manually excised. The DNA was purified using the QIAquick Gel Extraction Kit (Qiagen, Germany). The library quality and concentration were assessed and determined using the 2100 Bioanalyzer (Agilent, USA) and High Sensitivity DNA Kit (Agilent, USA). The sequencing template was prepared using the Ion OneTouch^TM 200 Template Kit V2 DL (Life Technologies, USA) according to the manufacturer’s protocol. The Ion Sphere Particles were enriched using Dynabeads MyOne Streptavidin C1 beads (Invitrogen, Life Technologies, USA). The efficiency of the enrichment process was assessed using the Ion Sphere Quality Control Kit (Life Technologies, USA). Genome sequencing was undertaken using the Ion Torrent PGM sequencer (Life Technologies, USA).