smRNA-seq of Homo sapiens: Exosomal MicroRNA
Total RNA isolated from each sample was used to construct sequencing libraries with the SMARTer smRNA-Seq Kit for Illumina (Takara Bio, San Jose, CA, USA), following the manufacturer's protocol. Briefly, input RNA was first polyadenylated to provide a priming sequence for an oligo-(dT) primer. cDNA synthesis was primed by the 3-smRNA dT primer, which incorporated an adapter sequence at the 5-end of each first-strand cDNA molecule. When the MMLV-derived PrimeScript Reverse Transcriptase reached the 5-end of each RNA template, it added non-templated nucleotides that were bound by the SMRT smRNA Oligo-enhanced with locked nucleic acid technology for greater sensi-tivity. In the template-switching step, PrimeScript Reverse Transcriptase used the SMART smRNA Oligo as a template for the addition of a second adapter sequence to the 3-end of each first-strand cDNA molecule. In the next step, full-length Illumina adapters (including index sequences for sample multiplexing) were added during the polymerase chain reaction (PCR) amplification. The forward PCR primer was bound to the sequence added by the SMART smRNA Oligo, while the reverse PCR primer was bound to the se-quence added by the 3-smRNA dT primer. The resulting cDNA molecule library included sequences required for clustering on an Illumina flow cell. The libraries were validated by checking their size, purity, and concentration on an Agilent Bioanalyzer (Agilent Tech-nologies, Santa Clara, CA, USA). The libraries were pooled in equimolar amounts, and sequenced on an Illumina HiSeq 2500 instrument to generate 51 base reads. Image de-composition and quality value calculations were performed using the Illumina pipeline modules.