RNA-Seq of lactobacillus bulgaricus:Control_1
Specifically, it includes the following steps: (a) Removement of rRNA: separating mRNA from total RNA for analyzing transcriptome information. (b) Fragmentation of mRNA: The enriched mRNA was randomly fragmented into small segments of approximately 200 bp by adding fragmentation buffer. (c) cDNA Synthesis: Using reverse transcriptase and random primers, single-stranded cDNA was synthesized from mRNA templates. During second-strand synthesis, dUTP was incorporated instead of dTTP in the dNTP mix, resulting in cDNA containing A/U/C/G bases. (d) Adapter Ligation: The End Repair Mix was added to convert the sticky-end double-stranded cDNA into blunt-end fragments. Subsequently, an adenine base was added to the 3' end to facilitate the ligation of Y-shaped adapters. (e) UNG Enzyme Digestion: The cDNA second strand was selectively digested using UNG enzyme, resulting in a library exclusively containing first-strand cDNA molecules. (f) Illumina HiSeq Sequencing: The library was amplified through 15 PCR cycles for enrichment, followed by quantification using TBS380 (Picogreen). Libraries were pooled in appropriate ratios and loaded onto the flow cell for bridge PCR amplification on the cBot system to generate clusters. Finally, paired-end sequencing (2  150 bp/300 bp) was performed on the Illumina NovaSeq X plus platform.