Pe adaptor oligonucleotides

Wild-type PA14 and the ΔpvrA mutant were cultured in FA-M9 at 37°C and harvested at the log phase (OD₆₀₀ = 1.0). Total RNA was isolated with an RNAprep pure bacteria kit (Tiangen Biotech, Beijing, China). Transcriptome sequencing was performed by Azenta Life Sciences (Suzhou, China). A Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA, USA) was used to selectively remove rRNA. The ribosomal-depleted mRNA was then fragmented and reverse-transcribed. First-strand cDNA was synthesized using ProtoScript II Reverse Transcriptase with random primers and actinomycin D. The second-strand cDNA was synthesized using the Second Strand Synthesis Enzyme Mix (include dACGTP/dUTP). The double-stranded DNA was purified by the AxyPrep Mag PCR Clean-up (Axygen) and then treated with End Prep Enzyme Mix for both end repair and addition of a dA-tailing in one reaction, followed by a T–A ligation to add adaptors to both ends. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adaptor oligonucleotides were ligated to prepare for hybridization. DNA fragments 300 bp in length with adaptor molecules ligated on both ends were enriched using the Illumina PCR Primer Cocktail. The library was then sequenced on a NextSeq 500 platform (Illumina). The sequence reads were mapped onto the PA14 reference genome (NC_002516.2)