Chunk #9 — Materials and methods — RNA-seq analysis of miRNA and mRNA transcriptomic changes in eight brain regions of AUD subjects (192 Set 1 RNA samples)
miRNA and mRNA expression profiles of the 192 selected Set 1 RNA samples were analyzed, respectively, by small RNA-seq and ribosome RNA (rRNA) depletion RNA-seq. Small RNA-seq was conducted as described in our previous study [28]. Briefly, small RNA-seq libraries were generated using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (Set 1) (NEB, Ipswich, MA, USA) with 250 ng of total RNAs. Purified cDNA libraries were pooled in equimolar ratios (12 libraries per pool) and multiplex sequenced at 1 × 75 bp on an Illumina HiSeq 2500 Sequencing System (Illumina, CA, USA). The Comprehensive Analysis Pipeline for miRNA Sequencing Data (CAP-miRseq) workflow [29] was used for raw reads (in fastq files) pre-processing, alignment, mature/precursor/novel miRNA qualification, and prediction. The mean total number of reads per sample was 16,591,602, and the mean mapping rate (aligned reads/reads sent to Aligner) was 73.2%. Principal component analysis (PCA) of miRNA transcriptome data of these 192 samples (from 8 brain regions) showed clustered CRB and VTA samples, but samples from six other brain regions could not be separated by brain regions using
