Small RNA-seq analysis of hypertensive patients with or without urinary albumin excretion
Plasma and urinary exosomal smaples were obtained by ultracentrifugation  then total RNA were extracted from exosomal and plasma samples and stored at -80C until library preparation. Individual samples were analyzed by random sequencing of small RNAs. Single patient libraries were prepared from 2 L of total RNA from each condition (total plasma urine exosomes or plasma exosomes) using CleanTag Small RNA library preparation kit (TriLink Biotechnologies US) adapting manufacturers protocol to very low input samples (Shore 2016). Briefly after adapter ligation and cDNA amplification containing 1 of 48 index sequences libraries were size-selected (138155 bp fragments) on a 15% Novex TBE PAGE gel (Life Technologies US) purified re-amplified 10 cycles quantified by RT-qPCR and pooled for multiplexed sequencing. Pools were normalized to 2 nM and sequenced on the HiSeq 2000 platform (Illumina US) with a 50-cycle single read mode (CNAG Barcelona Spain).