Onetouch 2 es station

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA integrities and qualities were assessed by Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and agarose gel electrophoresis. The RNA integrity number (RIN) of all samples was more than 7.0. Then, RNA samples from IM, CM, and CMT groups (n = 4 per group) were pooled with equal quantities, respectively, and were used for cDNA library construction and Ion-proton sequencing. Briefly, cDNA libraries for single-end sequencing were prepared using the Ion Total RNA-Seq Kit v2.0 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. After diluting and mixing of samples, the mixture was processed on OneTouch 2 instrument and was enriched on OneTouch 2 ES station (Life Technologies, Carlsbad, CA, USA). Finally, the enriched mixture samples were loaded on to 1 P1v2 Proton Chip and sequenced on Proton Sequencers according to the Ion PI Sequencing 200 Kit v2.0 (Life Technologies, Carlsbad, CA, USA).

The complementary DNA (cDNA) libraries for single-end sequencing were prepared using the Ion Total RNA-Seq Kit v2.0 (Life Technologies). The cDNA library was size selected by PAGE gel electrophoresis for miRNA sequencing. The cDNA libraries were then processed for proton sequencing. After diluting and mixing the samples, the mixture was processed on a OneTouch 2 instrument (Life Technologies) and enriched on a OneTouch 2 ES station (Life Technologies) to prepare the template-positive Ion PI™ Ion Sphere™ Particles (Life Technologies) according to the Ion PI™ Template OT2 200 Kit v2.0 (Life Technologies). After enrichment, the mixed template-positive Ion PI™ Ion Sphere™ Particles in the samples were loaded onto a P1v2 Proton Chip (Life Technologies) and sequenced on a proton sequencer according to the Ion PI Sequencing 200 Kit v2.0 (Life Technologies).

Total RNA of each sample was extracted with the Qiagen Rneasy Mini Kit and digested with Dnase I according to the manufacturer’s instructions (Qiagen, Hilden, Germany). The integrity and size distribution of the RNA were verified with Agilent 2200 Bioanalyser (Agilent Technologies., USA.). Samples with the RNA Integrity Number ≥8.0 were used for cDNA library construction. The cDNA libraries for single-ending sequencing were constructed using Ion Total RNA-Seq Kit v2.0 (Life Technologies, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. The cDNA libraries were then processed for the Proton Sequencing process according to the commercially available protocols. Samples were diluted and mixed, and the mixture was processed on a OneTouch 2 instrument (Life Technologies) and enriched on a OneTouch 2 ES station (Life Technologies) for preparing the template-positive Ion PI™ Ion Sphere™ Particles (Life Technologies) according to Ion PI™ Template OT2 200 Kit v2.0 (Life Technologies). After enrichment, the mixed template-positive Ion PI™ Ion Sphere™ Particles of samples was loaded on to 1 P1v2 Proton Chip (Life Technologies) and sequenced on Proton Sequencers according to Ion PI Sequencing 200 Kit v2.0 (Life Technologies).