Serum Small RNA Sequencing Protocol

We isolated total RNA of 461 subjects from serum samples using Qiagen miRNeasy Serum/Plasma extraction kit and QIAcube automation. All samples were quantified using the Nanodrop spectrophotometer prior to plating with the RNA concentration 30.97 ± 20.97 ng/μl (see Additional file 1: Fig. S1). We built the small RNA-Seq libraries by Norgen Biotek Small RNA Library Prep Kit and sequenced on the Illumina NextSeq 500 platform at 51 bp single end reads. The sequencing data was deposited in the Gene Expression Omnibus with the accession number GSE134897 [13 (link)]. COMPSRA was employed to evaluate the read quality and trim adapters [22 (link)]. Sequencing reads with quality score lower than 20 were removed. The qualified reads were aligned to human genome hg38 by STAR (v2.7.10b) [23 (link)] and miRNAs were annotated by COMPSRA on the basis of miRbase [24 (link)].