Agilent 2100 bioanalizer

A sequencing library was constructed with 100 ng of the equimolar pool by the amplicon fusion method (Ion Plus Fragment Library Kit, MAN0006846, Life Technologies). The library was quantified with the Agilent 2100 Bioanalizer (Agilent Technologies Inc, Palo Alto, California) prior to clonal amplification, and emulsion PCRs were carried out applying the Ion PGM Template OT2 400 kit as described in the user-guide (MAN0007218, Revision 3.0 Lifetechnologies) provided by the manufacturer. The library was sequenced in an Ion 318 Chip v2 on a Personal Genome Machine (PGM IonTorrent, Lifetechnologies) at Lifesequencing S.L (Lifesequencing, Valencia, Spain), using the Ion PGM Sequencing 400 kit following the manufacturer’s protocol (Publication Number MAN0007242, Revision 2.0, Lifetechnologies). Raw sequences obtained from the sequencing centre were processed with the MOTHUR software28 (link). A summary of sequencing statistics is available in Supplementary Table 1. Short (<100 bp) and low quality (

Peripheral blood samples were collected in PAXgene blood RNA tubes (PreAnalytix, Switzerland). PAXgene tubes were frozen using a sequential freezing process. This involves storing tubes at room temperature for 3 h, transferring to 4 °C overnight, followed by 6–8 h at −20 °C and then final storage at −80 °C. Total RNA (including the miRNA fraction) was isolated from whole-blood using the PAXgene Blood miRNA Kit (Qiagen, Canada), according to manufacturer’s instructions. Furthermore, total RNA was isolated from frozen brain, heart and liver tissues using the miRNeasy Mini Kit protocol (Qiagen, Canada) with no modifications. RNA and miRNA yield and quality were determined using the Nanodrop 1000 (Thermo Scientific, USA) and Agilent 2100 Bioanalizer (Agilent Technologies, USA).