Transcriptional Analysis of Lactobacillus fermentum

Bacterial RNA was extracted using a ZymoBIOMICS RNA Miniprep Kit (Zymo Research). Sequencing and library construction were performed on an Illumina HiSeq 2500 with 101-bp paired-ends. Ribosomal RNA was removed using a Ribo-Zero rRNA Removal Kit (Bacteria) (Epicentre). Libraries were prepared with a TruSeq RNA Sample Prep kit v2 (Illumina). RNA-sequenced reads were mapped to the L. fermentum genome (NCBI, NC010610.1) using STAR with alignIntron MAX 1^{25 (link)}. Then, the mapped reads were used to calculate read counts of genes using cufflinks^{29 (link)}, and the gene list was input into the Cytoscape plug-in ClueGO v2.5.4^{30 (link)} to annotate functionally grouped networks. Functionally related GO terms for biological processes in Escherichia coli (version: 18 November 2016) were grouped based on a kappa score greater than 0.4 with a network specificity of 4–10. Statistical significance was calculated using two-sided hypergeometric tests, and the false discovery rate was corrected using the Bonferroni step down method.