Superscript double stranded cdna synthesis kit

5 μg of RNA was taken from each sample, and an RNA library was constructed using an Illumina TruSeqTM RNA sample preparation kit. The messenger RNA (mRNA) was enriched and randomly broke into small fragments (about 300 bp). Subsequently, an Invitrogen SuperScript double-stranded cDNA synthesis kit and Illumina random primers were used to synthesize double-stranded cDNA. The target library was PCR amplified using Phusion DNA polymerase (NEB), after which PCR products were separated by electrophoresis on 2% agarose gels. Target bands were gel-extracted and the recovered cDNA libraries were quantified by TBS380 (Picogreen) and subsequently sequenced using an Illumina HiSeq 4000.

Total RNAs were isolated using TRIzol® reagent (Invitrogen, Waltham, MA, USA), and contaminating genomic DNA was removed using DNAase I (Takara, Otsu, Japan). The RNA quality was determined with a 2100 Bioanalyzer (Agilent, alo Alto, CA, USA) and quantified with ND-2000 (NanoDrop Technologies, Wilmington, DE, USA). Oligo (dT)-enriched mRNA was fragmented by fragmentation buffer, and the cleaved RNA fragments were reverse transcribed to establish the final cDNA library using a SuperScript double-stranded cDNA synthesis kit with random hexamer primers (Illumina, San Diego, CA, USA). After being end-repaired, adenylated, and ligated to the sequencing adaptors, the RNA-seq sequencing library was sequenced on an Illumina Hiseq xten/NovaSeq 6000 sequencer (Illumina, San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). The data were analyzed using the online platform of Majorbio Cloud Platform (www.majorbio.com, accessed on 5 December 2021). Statistical significance of the differentially expressed data was defined with p adjust < 0.05 and fold change ≥ 2.

An RNA-seq transcriptome library with 2 μg of total RNA was performed using Illumina's TruSeqTM RNA sample preparation kit (San Diego, CA). Then, random hexamer primers (Illumina) were used to synthesize double-stranded cDNA using a SuperScript double-stranded cDNA synthesis kit (Illumina). The library was sequenced by the Illumina HiSeq × 10 (2 × 150 bp read length) after being quantified by TBS380 and processed by Illumina GA Pipeline (version 1.6), yielding 150 bp paired-end reads. The reads were aligned to the E. coli K12 strain (NCBI reference sequence: NC_000913.3). XLSTAT software (2015 version, Addinsoft) was used for the principal-component analysis (PCA).