RNA-Seq of Bifidobacterium longum 1898B
Cells were inoculated with an OD600nm of 0.1. After inoculum, growth was monitored and at an OD600nm between 0.6 and 0.8 (exponential phase) cells were harvested by centrifugation at 6000rpm for 5 min. Growth assays were carried out in triplicate. Total RNA was isolated from cultures grown in the presence of sublethal concentration (sub-MIC), i.e., 4g/mL of antibiotic (AMC) in MRS w/o glucose at 2% (wt/vol) of galactose or MRS w/o glucose at 2% (wt/vol) of galactose (reference condition) and in MRS (reference condition for glucose) using previously described methods (15). Briefly, cell pellets were resuspended in 1 ml of QUIAZOL (Qiagen, United Kingdom) and placed in a tube containing 0.8 g of glass beads (diameter, 106 m; Sigma). The cells were lysed by shaking the mix on a BioSpec homogenizer at 4C for 2 min (maximum setting). The mixture then was centrifuged at 12,000 rpm for 15 min, and the RNA-containing sample was recovered from the upper phase. The RNA sample was further purified by using the RNeasy mini kit (Qiagen) as reported in the manufacturers instructions. The quality of the RNA was verified employing a Tape station 2200 (Agilent Technologies, USA). RNA amount was evaluated using a spectrophotometer (Eppendorf, Germany). For RNA sequencing (RNA-Seq), from 100 ng to 1 g of extracted RNA was treated to remove rRNA by using the QIAseq FastSelect  5S/16S/23S following the manufacturers instructions (Qiagen, Germany). The yield of rRNA depletion was checked by using a Tape station 2200 (Agilent Technologies, USA). Subsequently, a whole transcriptome library was constructed using the TruSeq Standard mRNA Sample preparation kit (Illumina, San Diego, USA). Samples were loaded into a NextSeq high-output v2.5 kit (150 cycles, single end) (Illumina) according to the technical support guide.