Nexteraxt library preparation protocol

Genomic DNA was extracted from fresh cultures of each isolate using the Isolate II Genomic DNA Kit (Bioline, London, UK), followed by quantification in the Qubit fluorometer (Invitrogen, Waltham, MA, USA) with the dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. The DNA was subjected to the NexteraXT library preparation protocol (Illumina, San Diego, CA, USA) prior to cluster generation and paired-end sequencing (2 × 150 bp) on a NextSeq 550 instrument (Illumina), according to the manufacturer’s instructions. FastQC v0.11.5 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (accessed on 16 February 2023)) was used for quality control and Trimmomatic v0.38 [25 (link)] for trimming low-quality bases.

Taking into account the sample type, location, and source, L. monocytogenes isolates from all matrices underwent WGS analysis. In the case of stillborn pools, only two out of the four strains were selected, as all isolates displayed identical antimicrobial phenotypes. The extraction of genomic DNA was carried out using the ISOLATE II Genomic DNA kit (Bio-line, London, UK) and quantified with the Qubit fluorometer (Invitrogen, Waltham, MA, USA) using the dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s guidelines. The NexteraXT library preparation protocol (Illumina, San Diego, CA, USA) was applied, and paired-end sequencing (2 × 150 bp) was performed on the NextSeq 2000 instrument (Illumina) following the manufacturer’s instructions.