Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: size fractionation
Layout: SINGLE
Construction protocol: TRIZOL LS reagent (Invitrogen) was used to extract total RNA from ~1.5 ml of serum, as described in the manufacturer’s protocol. RNA pellet was dissolved in 10 μl of RNase-free water, and subjected to library construction and deep sequencing. Each serum sample was independently subjected to library preparation and deep sequencing. All small RNAs of 15–52 nts were selected and sequenced using the Solexa system, following the manufacturer’s protocol (Illumina Inc., San Diego, CA). Library preparation, as well as cluster generation and deep sequencing, was performed according to the 5' ligation-dependent (5′ monophosphate-dependent) manufacturer’s protocol (Digital Gene Expression for small RNA; Illumina). For each sample, 5 μl of total RNA extracted from serum was used for small RNA library preparation. Small RNAs were size-selected between 17 and 52 nt according to the single-stranded DNA marker in the small RNA sequencing kit (Illumina). The library was quantified using picoGreen and qPCR. Sequencing was performed on a Genome Analyzer IIx (Illumina), and image processing and base calling were conducted using Illumina’s pipeline.