Phenol water

The cultures were centrifuged at 8,000 × g to precipitate bacterial cells. Total RNA was extracted using the hot phenol method as previously described with modifications (11 (link)). Subsequently, the bacterial cells were washed two times with RNAse-free saline or phosphate-buffered saline (PBS; cat. no. E607016-0500; BBI solutions, Cardiff, UK). Then, 400–600 µl TES solution was added according to the precipitation amount, and the bacterial cells were resuspended. The same amount of phenol-water (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was added followed by violent mixing. Centrifuge tubes containing a mixture of each sample, TES, and phenol-water were agitated at 65°C for 30–60 min in a Thermomixer Compact 5350 (Eppendorf, Hamburg, Germany), and then, the tubes were placed on ice and allowed to stand for 5 min. Then, the mixtures were centrifuged at 11,000 × g for 10 min at 4°C. The upper aqueous phase was selected and transferred to a new tube. Subsequently, a 1/2 volume of TRK-1002 lysis-solution and 2/3 volume of 95% ethyl alcohol was added to the upper aqueous phase, followed by vortex blending. Total RNA was then extracted using a TRK-1002 Purification kit (LC Sciences, Houston TX, USA), following the manufacturer's instructions. RNA quality was evaluated using an Agilent Bioanalyser (Agilent Technologies, Inc., Santa Clara, CA, USA).

The bacterial collection was added with phenol water (Sinopharm Chemical Reagent Co., Shanghai, China) and shaken violently to be mixed. The centrifuge tube was incubated at 65°C with an oscillating metal bath at maximum speed for 30–60 minutes, cooled on ice for 5 minutes, then centrifuged at 4°C for 10 minutes. The upper water phage was absorbed and added with 1/2 volume of TRK-1002 lysate (LC-Bio Technology CO., Ltd, Hangzhou, China). The concentration and purity of RNA were measured using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). An RNA integrity number (RIN) >7.0 can be used to construct a cDNA library, and the cDNA library was prepared and sequenced by Lianchuan Bio (Hangzhou, China). Ribosomal RNA were removed from the total RNA by using a Ribo-Zero Magnetic Kit (Epicentre, Madison, WI, USA), and then the library was generated using an Illumina Truseq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s recommendation. After digested into 200 nt fragments, double-stranded cDNAs were synthesized using uracil-N-glycosylase (UNG) to a cDNA cluster. The cDNAs were used to create the RNA-seq library by following the standard Illumina protocol (Lianchuan Bio, Hangzhou, China).