Ion torrent sequencing

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).

Genomic DNA from the whole-blood samples was extracted by a commercially available kit (Chemagen, Germany) at the Karolinska institute biobank or by QIAamp DNA Blood Maxi Kit (Qiagen, Germany). All selected exons, including at least 25 nucleotides surrounding the exonic regions, were amplified either using Agilent SureSelect custom design kit or Life Technologies AmpliSeq sequence enrichment method. The amplified DNA was analyzed by Illumina MiSeq or Life Technologies IonTorrent sequencing, respectively. The average target coverage was over 97% for both methods. Targeted exome sequencing was performed at the Uppsala Genome Center using standardized protocols provided by the manufacturer.

RNA quality was assayed on Agilent Tape Station 2200 using RNA HS screen tape assay. RNA (25 ng) was used to prepare a small RNA library using Clean Tag Small RNA library kit (TriLink biotech) following manufacturer recommended conditions. Adapter ligated RNA was reverse transcribed using Ion Torrent specific primer. cDNA was then amplified using Ion convert barcode primers containing a unique index to allow libraries to be pooled during Ion Torrent sequencing (Life Technologies). Library quality control (QC) was assayed on Agilent Tape Station using D1000 screen tape. Equally-pooled libraries (pmol) were clonally amplified onto Ion Sphere Particles (ISPs) supplied by Ion Pi HiQ OT2 kit. ISPs were then loaded onto Ion PI chip and sequenced using Ion Torrent Proton Sequencer.