Rneasy plus

RNA expression was measured by quantitative real-time PCR (qPCR) on a CFX384 thermocycler (Bio-Rad). RNA was collected by TRIzol Reagent (Life Technologies) or by column purification with RNeasy PLUS (Qiagen) or NucleoSpin RNA II (CloneTech) Kits according to manufacturer’s recommendation. Total RNA (50 ng µL⁻¹ final concentration) was used to generate first strand complimentary DNA (cDNA) using qScript cDNA Supermix (Quanta). For amplification, cDNA (0.5 µL per well), primers (0.4 µM final concentration), and SensiFAST Hi-ROX qPCR Master Mix (Bioline) were mixed and dispensed 5 µL per replicate, 4 replicates in a 384-well qPCR plate. Thermal cycler parameters: initial denaturation, 95 °C for 3 min followed by 40 cycles of annealing and extension, 60 °C for 5 s and denaturation, 95 °C for 15 s. Primers were validated by melt curve analysis (60–95 °C, 5 min) and gel electrophoresis of products. Data were analyzed and statistical analyses performed using CFX Manager (Bio-Rad) and plotted with Microsoft Excel. All gene expression was normalized to the expression of an appropriate internal control gene (18 S, GAPDH, RPL13A, RPL37A, and TBP). QPCR primer sequences are provided in Supplementary Table 1.

Bacterial cells pelleted following treatment with RNAprotect were resuspended in 100 μl of 2 mg ml^− 1 lysozyme solution and incubated for 10 min at room temperature. RNA was isolated using an RNeasy Plus (Qiagen) kit as per the manufacturer's instructions, except that the bacterial lysate was aspirated through a 22 gauge needle once RLT buffer had been added. Total RNA samples were then treated with DNase I (Ambion) for 1 h and concentrated using an RNeasy MinElute Cleanup kit (Qiagen). RNA concentration was determined by using a NanoDrop spectrophotometer (Thermo Scientific) and RNA integrity verified by an Agilent Bioanalyzer. Approximately 10 μg of total RNA was converted into cDNA using random hexamers and SuperScript III (Life Technologies) as per the manufacturer's instructions.
Samples were amplified using oligonucleotide primers for crr (y1485), y1667, y0666, y3909, y3519, y2882 (psaA), y1918 and the IQ SYBR Green Supermix (Bio-Rad) via a two-step protocol on a Bio-Rad CFX384 real-time system. Oligonucleotide sequences are tabulated in Table S1. Cycling parameters were optimized using Y. pestis KIM6+ genomic DNA. Expression levels of the genes of interest were determined as a ratio to the levels of the constitutively expressed gene crr (Vadyvaloo et al., 2010 (link)).

Total RNA was extracted from each cell using RNeasy Plus (QIAGEN) after 168 h siRNA transfection, and the quantity and quality was determined using Bioanalyzer (Agilent Technologies). Single read sequencing was performed with NextSeq 500 (Illumina) using 75 cycles High Output kit v2. Sequences were trimmed for sequencing adapters and low quality 3' bases using Trimmomatic version 0.35 [3 (link)] and aligned to the reference Chlorocebus sabaeus genome version ChlSab1.1 (gene annotation from Ensembl 104) or Homo sapiens genome version GRCh38 (gene annotation from Gencode version 37) using STAR version 2.7.1a [7 (link)].
Raw gene expression counts were obtained directly from STAR, and normalization and differential expression analysis were performed using DESeq2 version 1.30.1 [24 (link)]. Metascape software was used for gene ontology analysis [56 (link)]. We used unadjusted p-values for U2OS analysis as false discovery rate calculation has low power in datasets with small effect sizes (i.e. low fraction of changed genes in RNAseq) [22 (link)], and in our dataset Benjamini–Hochberg procedure resulted in over-represented p.adjusted values (Additional file 2: Table S1). For comparison analyses between COS-7 and U2OS, gene identifiers were converted from C. sabaeus to H. sapiens using the R/Bioconductor package biomaRt (version 2.48.3).