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 (NZ_AP012324.1) using STAR with alignIntron MAX 1 [19 (link)]. Then, the mapped reads were used to calculate read counts of genes using cufflinks [23 (link)], and the gene list was inputted into Cytoscape plug-in ClueGO v2.5.4 [22 (link)] 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.
Free full text: Click here
