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.