Magnetic bead purification module

Small RNA library preparation was performed using the Ion Total RNA-Seq kit v2 (Life Technologies) for both EV samples. For each individual library, RNA was ligated to adapters containing a unique index barcode (Ion Xpress™ RNA-Seq Barcode 1–16 Kit, Life Technologies) to allow libraries to be pooled during Ion-Torrent sequencing (Life Technologies). All libraries were constructed according to manufacturer’s protocol. Briefly, RNA samples were reverse transcribed to cDNA using adapter-specific primers. Using the Magnetic Bead Purification Module (Life Technologies), cDNA samples were size-selected from 94 to 200 nt (the length of the small RNA insert including the 3′ and 5′ adapters). PCR amplification was then performed followed by a library clean-up step using nucleic acid beads (Life Technologies). The quality and quantity of each library were determined by Agilent 2100 Bioanalyser using High Sensitivity DNA kit (Agilent Technologies). Equally pooled libraries were clonally amplified onto Ion Sphere™ Particles (ISPs) supplied by the Ion PGM™ Template OT2 200 kit (Life Technologies). ISP templates were produced by using the OneTouch™ 2 Instrument and enrichment system (Life Technologies). ISPs loaded with libraries were sequenced on the Ion-Torrent PGM™ using Ion™ 318 v2 chips (Life Technologies) and the Ion PGM™ 200 Sequencing Kit v2 (Life Technologies).

The miRNAs libraries were prepared from the exosomal RNA using Xpress RNA-seq Barcode 1–16 Kit, Life Technologies, Carlsbad, CA, USA). For each library, 50 ng of RNA was used for ligation adapters with unique index barcodes. Furthermore, RNA samples were reverse transcribed to cDNA using adaptor-specific primers designed for small RNA sequencing. cDNA samples were then used for the size-selection step. Size selection from 94 to 200 nt that included the adaptor sequences around 25 bp was performed using Magnetic Bead Purification Module (Life Technologies). The yield and size distribution of the contracted small RNA libraries were verified using the Agilent 2100 Bioanalyzer instrument with the High Sensitivity DNA chip from Agilent. Only libraries with good quality were equally pooled. They were clonal amplified onto Ion Sphere Particles (ISPs) supplied by the Ion OneTouch 200 Template Kit v2 DL kit (Life Technologies) and enriched using the One Touch 2 ES System (Life Technologies).