RNA-seq Library Preparation and Analysis

RNA extraction was performed with a PAXgene Blood RNA kit IVD (QIAGEN, Hilden, Germany). The RNA-seq library was prepared with a TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA, USA). The messenger RNA libraries were sequenced by paired-end 100 cycles on the HiSeq 2500 (Illumina). Low-quality bases in the raw reads were filtered out by FastQC ver 0.11.5 [16 ] and potentially existing sequencing adapters were trimmed with Skewer ver 0.2.2 [17 (link)]. The high-quality reads were mapped to the human reference genome hg19 (downloaded from UCSC genome browser, https://genome.ucsc.edu) by STAR ver 2.6 [18 (link)]. The gene expression level was quantified based on the aligned reads by Cufflinks package ver 2.2.1 [19 (link)]. The gene annotation of the reference genome was used as gene models and the expression values were calculated in Fragments Per Kilobase of transcript per Million fragments mapped (FPKM) unit.