Rnaprotect bacteria reagent kit

Each strain was cultured in BHI broth for 12 h after inoculation. The cultures were then pelleted by centrifugation (1,600g, 15 min, 4°C), and the bacterial RNA was protected using an RNAprotect Bacteria Reagent Kit (Qiagen, Tokyo, Japan), and isolated using the RNeasy Mini kit (Qiagen) as previously described 32. TURBO DNA‐free Kit (Thermo Fisher Scientific, Yokohama, Japan) was used to remove any contaminating DNA. Then, the RNA was reverse transcribed into cDNA using High‐Capacity cDNA Reverse Transcription Kits (Thermo Fisher Scientific) according to the manufacturer's instructions.
The PCRs for the ΔΔCT method were performed as follows: 95°C for 20 s, followed by 40 cycles of 30 s at 95°C and 30 s at 60°C. The 16S rRNA housekeeping gene served as an endogenous control to normalize the expressional levels between the samples. The DNA sequences of the primers are described in Sup Table S2 23, 24, 27, 33, 34, 35, 36, 37.

Triplicate cultures of T6⁺ and T6⁻A. baumannii ATCC 17978 were grown overnight, diluted into fresh medium, and grown to an optical density at 600 nm (OD₆₀₀) of 0.5. Three hundred microliters of cells was lysed, and RNA was stabilized by using the protocols and buffers for the RNAprotect Bacteria Reagent kit (Qiagen). RNA was purified with the RNeasy minikit (Qiagen), and rRNA was depleted with the RiboZero kit (Illumina). Directional RNA-seq libraries were constructed with the PrepX RNA-Seq for Illumina library kit (Wafergen) and sequenced with an Illumina NextSeq 500 as paired-end 75-base reads. Reads were mapped as paired ends to the A. baumannii ATCC 17978-mff genome, raw counts and TPM values were calculated, and differentially expressed genes were identified with the DESeq2 package in R (74 (link)).

Microarray experiments with drugs in M. tuberculosis were performed according to previously reported methods [19 (link)]. First, RNA was extracted from M. tuberculosis with an RNAprotect Bacteria Reagent kit (QIAGEN, Hilden, Germany). The extracted RNA was sent to ebiogen (Seoul, Korea) for microarray analysis. Briefly, RNA samples were reverse transcribed into cDNA, and target cRNA probes were synthesized and hybridized on a MYcroarray.com (accessed on 08 September 2022) (M. tuberculosis) 3 × 20 k Microarray. Images were then acquired with an Agilent DNA microarray scanner (Agilent, Technology, Santa Clara, CA, USA) and quantified using Feature Extraction software (Agilent Technology, Santa Clara, CA, USA). Differentially transcribed genes were sorted by functional category [20 (link)]. A hypergeometric distribution method was used to determine which specific functional category genes were affected by the drug [21 (link),22 (link)]. Genes identified with a fold change cutoff of >2 and p < 0.01 were analyzed for an in-depth study.