Chunk #11 — Materials and methods — RNA-seq analysis of miRNA and mRNA transcriptomic changes in eight brain regions of AUD subjects (192 Set 1 RNA samples)
Since most of the 192 selected postmortem brain RNA samples had RINs below 7 (Table S1), the KAPA RNA HyperPrep Kit with RiboErase (KAPA Biosystems, Wilmington, MA, USA) was used to deplete ribosomal RNAs (rRNAs) and construct RNA-seq libraries with 1 μg of total RNAs as the starting material. Pooled libraries were loaded into individual lanes (80 pooled libraries/lane) of the NovaSeq S4 flow cell (Illumina, San Diego, CA, USA) by running the NovaSeq Xp workflow for 100 bp paired-end sequencing on a NovaSeqTM 6000 system (Illumina, San Diego, CA, USA). The bulk RNA-seq processing pipeline Pipeliner [30] was utilized to quantitate gene and isoform expression. The mean total number of reads per sample was 38,758,477, and the mean mapping rate (aligned reads/reads sent to Aligner) was 84.6%. PCA plotting of the mRNA-seq data showed similar sample clustering patterns as above using the miRNA-seq data (Fig. S1b). The rRNA depletion RNA-seq fastq files and normalized read counts are available for downloading from the NCBI GEO database (accession number: GSE181982).
