Lysozyme solution

After cross-linking, cells were washed twice with PBS. Lysozyme solution (TIANGEN) and 10% SDS (Sigma) were added for cell lysis at 64 °C for 2 min. Lysates were cooled to 4 °C. RNA was extracted by the acid guanidinium thiocyanate-phenol-chloroform extraction method [28 (link)]. DNA contamination, if any, was eliminated using DNase I (NEB), which was deactivated by heating to 90 °C for 2 min.

The extraction of total RNA was carried out by following the manufacturer's instructions of a UNIQ‐10 column Trizol total RNA extraction kit (Sangon Biotech Co., Ltd.) and an iScript cDNA synthesis kit (Bio‐Rad Laboratories, Inc.), respectively. Before RNA extraction, 20 mg/ml lysozyme solution (Tiangen Biotech Co., Ltd.) was added to pellets and incubated at 37°C for 40 min to lyse the cells. The purity and concentration of the extracted RNA were checked using a microspectrophotometer (SMA4000, Merinton Instrument, Inc.), and the integrity of extracted RNA was measured by 1.5% agarose gel electrophoresis.
For the first‐strand cDNA synthesis, 800 ng of total RNA was used for reverse transcription according to the instructions of the iScript cDNA synthesis kit (Bio‐Rad Laboratories, Inc.). Real‐time qRT‐PCR was performed using a StepOnePlus Real‐Time PCR System (ABI) to evaluate the transcriptional levels of stress/virulence‐related genes. The specific primers of genes and associated functional descriptions were designed and synthesized by Sangon Biotech Co. Ltd., which are listed in Table 1. The relative expressions of these stress/virulence‐related genes in L. monocytogenes were calculated using the 2^−△△Ct method (Li et al., 2019 (link)).