2.7. Bacterial RNA Sequencing and Data Analysis

Bacterial RNA was extracted using the ZymoBIOMICS RNA Miniprep Kit (Zymo Research, Irvine, CA). Sequencing and library construction were performed on the Illumina Hiseq 2500 with 101 bp paired-end. Ribosomal RNA was removed using the Ribo-Zero™ rRNA Removal Kit (Bacteria) (Epicentre, Madison, WI). Libraries were prepared with the TruSeq RNA Sample Prep kit v2 (Illumina). RNA-sequenced reads were mapped on the reference genome of Bifidobacterium breve ({"type":"entrez-nucleotide","attrs":{"text":"NZ_AP012324.1","term_id":"844741909","term_text":"NZ_AP012324.1"}}NZ_AP012324.1) using STAR with alignIntron MAX 1 [19]. Then, the mapped reads were used to calculate read counts of genes using cufflinks [23], and the gene list was inputted into Cytoscape plug-in ClueGO v2.5.4 [22] to annotate functionally grouped networks. Functionally related GO terms for biological processes in Escherichia coli (version: November 18, 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.