Chunk #14 — Materials and methods — RNA-seq analysis of miRNA and mRNA transcriptomic changes in eight brain regions of AUD subjects (192 Set 1 RNA samples) — Microarray analysis of miRNA and mRNA transcriptomic changes in six brain regions of AUD subjects (96 Set 2 RNA samples)
For mRNA transcriptome analysis, the Affymetrix ClariomTM D human array (Affymetrix, Santa Clara, CA, USA) was used following the manufacturer’s instructions. This array allows interrogating more than 540,000 transcripts (including coding and long non-coding genes, exons, and alternative splicing events as well as rare transcripts) using over 6.7 million probes. About 500 ng of total RNA was used in the Affymetrix Clariom D human array assay. Probe cell intensity files (or CEL files) for transcripts were generated using the AGCC software. They were then analyzed using the Affymetrix EC software (v1.4.1) with the “Gene Level - RMA-Sketch (robust multi-array average with sketch quantile normalization)” workflow as the default analysis for background adjustment and signal normalization as well as log2 transformation to create probe-level summarization files (or CHP files). Quality control (QC) analysis of the CHP files was performed within the EC software, and the quality of the mRNA expression array data was visualized using box plots (Fig. S2b). The CHP files for case and control samples were further analyzed by statistical programs to identify differentially expressed mRNAs. The Affymetrix mRNA expression data has been deposited in the NCBI GEO database (accession number: GSE180722).
