RNA-sequencing

Overnight culture of Bi-26 in mBM58+ lactose was used as a 1% inoculum for 3 tubes of 10 mL of either mBM58+ 2′-FL or mBM58+ lactose. Lactose was used as the control samples. A600 was checked according to the growth curve and samples (one 10 mL tube) were taken during early phase (A600 = 0.25–0.30), mid log (A600 = 0.5–0.7), and late log (A600 = 0.9–1.1).

Immediately after each sample reached the growth phase, samples were centrifuged at 10,000 x g for 5 minutes, supernatant was removed, and the pellet was re-suspended in 1 mL of Trizol and frozen at −70 °C. The cell pellets were later thawed, transferred to a Lysing Matrix B 2 mL tube (MPBio, 116911050), and disrupted using a Bead ruptor elite (OMNI international, Georgia, United States) at 6.30 m/s for 2 minutes. The lysate was subjected to a chloroform organic extraction and followed by purification using RNeasy Mini Kit (Qiagen, Hilden, Germany, 74104).

Ribosomal RNA was removed prior to library construction using Ribo-Zero rRNA Removal Kit Gram-Positive Bacteria (Illumina, MRZGP126). Stranded cDNA libraries were prepared using TruSeq Stranded mRNA Kit (Illumina, 20020594), quantitated by Agilent TapeStation, pooled, and sequenced on one flowcell lane for 75 cycles using paired-end 75 basepair sequencing on Illumina 2500 HiSeq Rapid Cluster Kit (Illumina, V2, PE-402-4002) and HiSeq Rapid SBS Kit (Illumina, V2, FC-402-4021).

Paired-end reads were imported and mapped to the Bi-26 genome using the RNA mapping tool SeqMan Pro in DNAStar V12.3.1.4 (Madison, Wi) under the default settings. Processed using QSeq, and normalized by reads per kilobase of transcript per million mapped reads (RPKM).

Regression analyses of the RNA data were made in ArrayStar software V12.3.1. Statistical analyses between sets of samples were analyzed using DESeq 2 method in Geneious 11.0.4. Differences in expression were considered significant if the Absolute Confidence (−Log10 adjusted p-value) was +1.00, representing, and the Log₂ ratio was at +1.00, representing p < 0.05 and a >2x fold change, respectively, after normalization.