Novaseq pe150 platform

Total genomic DNA of these two “stealth” MRSA isolates and their revertants were extracted using the TaKaRa MiniBEST Bacteria GenomicDNA Extraction Kit (TaKaRa Bio Inc., Beijing, China). The whole genomes of wild-type “stealth” MRSA isolates and one revertant of the nasal swab strain were sequenced at the Beijing Genomics Institute using the long-reads PacBio third-generation sequencing method (Pacific Biosciences of California, Inc., Menlo Park, CA, USA) and the short-reads Illumina NovaSeq PE150 platform (Illumina, San Diego, CA, USA). After filtering low-quality reads, the clean data were obtained, which were preliminarily assembled using Link v5.0.1 and subsequently corrected with the Illumina data. Then, Rapid Annotation using the Subsystem Technology v2.0 server was used for gene annotation. Using BLAST, comparative genome analysis was performed among the parental and variant strains. The mutated genes were then confirmed using Sanger sequencing, and the mecA gene of revertants from the clinical strain was analyzed using conventional sequencing, as previously described by us (1 (link)). The sequences were aligned and further analyzed using the Qiagen CLC Genomics Workbench (33 ).

The crypts, villus and MSCs were isolated from mouse intestine respectively. Then RNA of these cells was extracted by RNeasy mini kit (74,104, Qiagen) as manuals. cDNA libraries were conducted with the Ovation RNA-Seq System V2 kit (NuGEN) and subjected to high-throughput sequencing on an Illumina Novaseq PE150 platform. RNA-seq was carried out with two biological replicates. Sequencing reads were aligned to mouse genome reference (mm10) using STAR (Spliced Transcripts Alignment to a Reference) with default parameter. Differential expression analysis was performed using the R package DESeq2 based on gene counts data. Differentially expressed genes were identified with Log₂FoldChange absolute value > 1 and p value < 0.01.

The Visium Spatial Gene Expression Slide & Reagent kit (10× Genomics) was used to construct sequencing libraries according to the Visium Spatial Gene Expression User Guide. A 10 μm frozen tissue section was placed on one of the Visium gene expression slide capture areas. After tissue hematoxylin and eosin (H&E) staining, bright‐field images were acquired as described in the spatial transcriptomics procedure. Tissue permeabilization was performed for 18 min, as established in the tissue optimization procedure. Then, reverse transcription was conducted and sequencing libraries were prepared following the manufacturer's protocol. Sequencing was performed with a NovaSeq PE150 platform according to the manufacturer's instructions (Illumina) at an average depth of 300 million read pairs per sample.

RNA sample quality was assessed using a Nanodrop (ThermoFisher) and Agilent 5400 (Agilent Technologies, Santa Clara, CA, USA). Prokaryotic mRNA sequencing was performed using the NovaSeq PE150 platform (Illumina, San Diego, CA, USA) at the Novogen facility (Sacramento, CA, USA). The library was prepared by a Ribo-Zero protocol (250 − 300 bp insert strand specific library with rRNA removal using NEB Ribo-Zero Magnetic Kit). Paired-end sequencing produced 150 bp reads, to a depth of ~2 G output per sample. Sequences were mapped to a reference genome, Fusobacterium nucleatum ATCC 23726 (GenBank accession: CP028109) using a Bowtie2 pipeline adjusted for paired-end sequencing. Differential gene expression was analyzed using the DEseq2 pipeline in Rstudio as previously described [25 ]. The total mapping rates with respect to the annotated genome for Fn 23726 were >98%. The false discovery rate (FDR) was set to 5% and genes with a log₂FoldChange of >1 or < −1 and a p-adjusted value (p-adj) < 0.001 were considered significant. All reported data are representative of three biological replicates.

NEBNext^® UltraTM DNA Library Prep Kit for Illumina (NEB, USA) was used for DNA extraction to construct sequencing libraries, following the manufacturer’s protocols. The qualified library was sequenced on the Illumina NovaSeq PE150 platform at Wekemo Tech Co., Ltd. Shenzhen, China.
Raw sequenced libraries were processed at Wekmo Tech Co. using Kneaddata software. First, sequencing adaptors, amino acid bases with quality scores less than 20, and sequences shorter than 50 bp were trimmed based on the Trimmomatic algorithm (Bolger et al., 2014 (link)). To reduce host pollution, genes of soil bacteria origin were deleted by aligning the sequenced data with bacterial genes on Bowtie2 (Langmead and Salzberg, 2012 (link)). Finally, clean data were obtained.

Total genomic DNA was extracted from 25 salivary samples using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.). DNA concentration and purity were determined using TBS-380 and NanoDrop2000 instruments. A sequencing library of 400 bp DNA fragments was constructed using a NEXTFLEX® Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA), and sequencing was performed on Illumina NovaSeq PE150 platform (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Metagenomic sequencing data have been published within the NCBI Sequence Read Archive database (SRP327008). After performing sequence quality control and removing any reads from the human genome, we assembled metagenomic data using MEGAHIT (https://github.com/voutcn/megahit, version 1.1.2) and selected contigs with lengths ≥ 300 bp for further gene prediction and the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analyses using Diamond (http://www.diamondsearch.org/index.php, version 0.8.35) alongside KEGG database (http://www.genome.jp/keeg/) with an e-value cutoff of 1e-5.

Sequencing was performed with a Novaseq PE150 platform according to the manufacturer’s instructions (Illumina) at an average depth of 300 million read-pairs per sample.