Bacterial total rna extraction kit

Total RNA of L. reuteri LR08 was extracted using a Bacterial Total RNA Extraction Kit (Tiangen Biotech Co., Ltd., Beijing, China). The RNA-seq transcriptome library was constructed using Illumina TruSeqTM RNA Sample Prep Kit. The mRNA was separated by oligo-dT magnetic beads. First, fragmentation was performed with fragment buffer, and then double-stranded cDNA was synthesized using SuperScript double-stranded cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). The target cDNA fragment of 200 bp–300 bp was selected after completing the repaired end of the synthesized cDNA. A total of 15 cycles of PCR amplification were conducted using Phusion DNA polymerase (NEB). The two-terminal RNA-seq sequencing library for computer sequencing was performed using Illumina Novaseq 6000 (2 × 150 bp) after TBS380 quantification.

The standard strain (ATCC11845) and the induced strain were scraped from the plate into the TSB liquid medium, and the OD was adjusted to 1 and cultured in the TSB liquid medium for 2 h, and the bacteria were collected. The total RNA was extracted using the bacterial total RNA extraction kit [Tiangen Biochemical Technology (Beijing) Co., Ltd., China], and the RNA purity was determined using NanoDrop 2000, and then sent to Lianchuan Biological Company for quality inspection. After passing the quality inspection, the transcriptome was detected. The raw data of transcriptome sequencing were filtered to obtain high-quality data information; DESeq v1.20.0 software was used to study the differentially expressed genes among the three groups. The conditions were set as the difference multiple |log₂FC| > 1, with a significance p-value of <0.05. The mRNA of the three samples was screened for differentially expressed genes.

For the transcriptome analysis, we only used the transcriptome datasets that were obtained at the exponential growth phase after induction. The samples were frozen immediately in liquid nitrogen and sent to Sangon Biotech (Shanghai, China) for transcriptome sequencing. Total RNA was extracted using the Bacterial Total RNA Extraction Kit (TIANGEN, Beijing, China). Sequencing was performed based on the Illumina Xten platform. Raw sequencing data were quality-controlled and mapped to the reference genomic sequences, and then the reads mapped to genes were counted. After calculating the expression of genes, differentially expressed genes (DEGs), clusters of orthologous groups (COGs), KEGG functional enrichment analysis, and KEGG pathway analysis were performed. DEGs were identified according to the following rules: a log2-fold change (FC) >2 and a p value < 0.05 [23 (link)].