RNA-Seq gene expression analysis and pathway enrichment analyses
To prepare RNA samples for RNA-seq, total RNA was isolated from human PBCMCs treated with 5 mM butyrate (or vehicle) for 24 h, using the RNeasy Micro Kit (74004, Qiagen). High-throughput sequencing was performed on the Illumina HiSeq 4000 sequencer. Reads were generated of 50 base-pairs in length and alignment was performed using HISAT (Hierarchical Indexing for Spliced Alignment of Transcripts). Tag directories were generated for each sample with removal of duplicate reads (-tbp 1 option). Quantification and normalization of the RNA-Seq data was performed using the open-source software HOMER 53. Differential expression was calculated using DESeq2 within the environment of HOMER. Significant differentially expressed genes were defined as differential genes with an adjusted P value < 0.05 (Wald test). To filter out significant differences among lowly expressed genes, an average RPKM value higher than 1 in at least one condition was required. The remaining genes all had a Log2 fold change higher than (-)0.8, we tolerated this cut-off for further downstream analyses. Pathway enrichment analysis was done using Metascape 54.