Total rna isolation system

Total RNA was extracted from 6 bacterial samples (O_S29_1, O_S29_2, O_S29_3, S29_1, S29_2, S29_3) separately by a Total RNA Isolation System (Promega) according to the manufacturer’s protocol. The quality of the RNA samples was examined using the Agilent 2100 Bioanalyzer. Library construction and Illumina sequencing was performed by Novogene China. An RNA-seq analysis was performed according to the protocol recommended by the manufacturer (Illumina Inc.) (Qiao et al., 2016 (link); Liu et al., 2019 (link)).
For data analysis, RNA-Seq reads were mapped to the reference genome of S. aureus Newman (NC_009641). Differentially expressed genes (DEGs) were identified by the edgeR package based on Genes with two criterions: |log2(fold change)| > 0.58499 (|fold change| >1.50003) and p < 0.05

Total RNA was extracted from ECPCs using RNAgents (Total RNA Isolation System, Promega, Madison, WI) and quantified spectrophotometrically. A volume of 500 ng of RNA was then retrotranscribed using ImProm-II reverse transcriptase (Promega, Madison, WI). Aliquots of 2 μl of the cDNA were used for PCR amplification. The specific primers used for the identification of human αv, β3, and GAPDH were designed according to published human cDNA sequences in the Genbank database, using FastPCR software [50 (link)]: αv (forward5′-CTA TGA GCT GAG AAA CAA TGG TCC-3′ and reverse 5′GCT GCT CCC TTT CTT GTT CTT C-3′690-bp product); β3 (5′- GGG GAC TGCC TGT GTG ACT C-3′ and reverse 5′-CTT TTC GGT CGT GGA TGG TG-3′ 610-bp product); GAPDH (forward: 5′-ACC ACA GTC CAT GCC ATC AC-3′ and reverse: 5′-TCC ACC ACC CTG TTG CTG TA-3′, 452-bp product). PCR was carried out on a Perkin Elmer Thermal cycler. Ten microliters of each PCR products were visualized after electrophoresis in a 2 % agarose. cDNA products were evaluated on the basis of a standard PCR marker (Promega) and quantified by densitometric analysis using ImageJ software (NIH).

The cells were seeded into an M6 well plate with 10000 cells per well. After treatment with EEROP in 72 hours, HeLa cells were trypsinized and centrifuged 300 × g for 5 minutes to get the cells pellet. The RNA of HeLa cells was subsequently extracted with a Total RNA Isolation System from Promega based on the manufacturer's protocol. The results of RNA extraction were evaluated with BioDrop at a 260 nm wavelength. The synthesis of cDNA in this study was carried out using the GoScript Reverse Transcription System from Promega using the manufacturer's instruction. PCR for cDNA was performed in the total volume of 20 μL with 5 minutes at 25°C for the priming process, 1 hour at 42°C for the reverse transcription process, and 15 minutes at 70°C for inactivating the reverse transcriptase enzyme.

To prepare RNA for transcriptome analysis, single bacterial colonies were picked and grown in 5 mL of NYG medium at 28 °C for 24 h at 200 rpm. These cells were transferred into 50 mL of NYG medium again at 28 °C for 24 h at 200 rpm. RNA was harvested once the cell turbidity had reached an OD₆₀₀ of 0.6, using a Total RNA Isolation System (Promega) according to the manufacturer’s protocol. Contaminating genomic DNA was removed using RNase‐free DNase I and verified by PCR. RNA quantity was initially determined by a Nanodrop spectrophotometer ND‐8000 (NanoDrop Technologies, Wilmington, DE, USA), and RNA quality was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies, CA, USA). Total RNA was sent to Novogene (Beijing, China) for library construction and strand‐specific RNA sequencing. Sequencing libraries were generated using a NEBNext Ultra™ Directional RNA Library Prep Kit for Illumina (New England BioLabs) following the manufacturer’s recommendations, and sequenced on an Illumina (CA, USA) HiSeq 2000 platform. Clean reads were mapped to the reference genome. To eliminate the influence of different gene length and sequencing discrepancy on the calculation of gene expression, the RPKM (reads per kilobase per million mapped reads) method was used to calculate the gene expression levels.

By using the Total RNA Isolation System (Promega), total RNA of six cell samples was extracted. The extracted RNAs quality was checked using Agilent 2100 Bioanalyzer. Library construction and Illumina sequencing was undertaken by Novogene China.
All RNA-seq reads were aligned to the genome of A. baumannii ATCC 17978 (https://www.ncbi.nlm.nih.gov/assembly/GCA_000015425.1; accessed on 1 March 2007) for data analysis. Only genes with an adjusted p-value  <  0.05 and |fold change|  >  1.5 were regarded as differentially expressed genes (DEGs).

The putative type II TA systems in the S. suis 2 strain SC84 were predicted with TAfinder (http://202.120.12.133/TAfinder/TAfinder.php). In our work, S. suis 2 was grown to the mid-log-phase and was used to extract the total RNA. The total RNA was purified by using an SV (spin or vacuum) total RNA isolation system (Promega, Madison, WI, USA), according to the manufacturer’s protocol. The RNA integrity and concentrations were determined by agarose gel electrophoresis and NanoDrop, respectively. The cDNAs were generated from these RNA samples with HiScript II Q RT SuperMix (Vazyme, Nanjing, China). We used the specific primers (0547F/0548R, 0791F/0790R, 0792F/0791R, 0842F/0841R, 0860F/0861R, 1035F/1034R, 1349F/1348R, and 1821F/1820R) in Table S2 to confirm the co-transcription of putative toxin genes and antitoxin genes.