Hiseq x 10 pe150 platform

Genomic DNA of S. aureus ST72 isolates was extracted using the Ezup Column Bacteria Genomic DNA purification kit (Sangon Biotech, Shanghai, China). Sequencing of the ST72 genomes was performed on a HiSeq X 10-PE150 platform (Illumina, San Diego, CA, USA). After adaptor trimming and quality filtering, the processed Illumina reads were assembled using SPAdes v3.14.1 (30 (link)) with default settings. SCCmec and spa typing were performed using the web-based SCCmecFinder (https://cge.cbs.dtu.dk/services/SCCmecFinder) and SpaFinder (https://cge.cbs.dtu.dk/services/spatyper), respectively. Potential open reading frames (ORFs) were predicted and annotated using Prokka v1.14.5 (31 (link)). Additional genomes of 83 ST72 isolates with available geographic information were downloaded from the NCBI genome database for comparison (Table S1).

Single-cell library preparation was performed using ulRNA-seq protocol [47 (link)]. First, RNA templates were denatured at 72 °C for 3 min, and immediately placed on ice afterward. Second, template switching first-strand cDNA synthesis was performed using a 5′-biotinylated TSO oligo. Third, PCR pre-amplification was performed directly after reverse transcription, and then the cDNA product was purified with 0.8 × Ampure XP beads. Fourth, cDNA concentration was measured using the Qubit dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Fifth, 1 ng of cDNA was used for the tagmentation reaction carried out with One-step DNA Lib Prep Kit for Illumina (ABclonal, Wuhan, China). Finally, cDNA library fragment distribution was detected with the Agilent 4200 High Sensitivity DNA Assay Kit (Agilent Technologies, Palo Alto, CA, USA). According to the detection results, the quality of the cDNA library was determined, and the subsequent cDNA library was sequenced on the Illumina HiSeq X10 PE150 platform (Illumina Inc., San Diego, CA, USA).

Genomic DNA was extracted from K. variicola strains Z5 and K6 using a QIAamp DNA kit (Qiagen). Whole-genome sequencing was performed using an Illumina HiSeq X10 PE150 platform. The average nucleotide identity (ANI) was calculated according to a previously established method (Goris et al., 2007 (link))². The sequence data were deposited at NCBI under the accession numbers JAAGKS000000000 and JAAGKT000000000.