Hiseq 3000 platform

Urban sewage was collected globally from 79 sample locations, covering seven geographical regions from 74 cities in 60 countries^{17 (link)}. DNA was extracted from the sewage pellets according to an optimized protocol using the QIAamp Fast DNA Stool Mini Kit including twice the input material and initial bead beating⁵⁷ . DNA from all samples was mechanically sheared to a targeted fragment size of 300 bp using ultrasonication (Covaris E220evolution). Library preparation was performed with the NEXTflex PCR-free Library Preparation Kit (Bioo Scientific). The Bioo NEXTflex-96 adapter set (Bioo Scientific) was used, and in batches of roughly 60 samples, the libraries were multiplexed and sequenced on the HiSeq. 3000 platform (Illumina), using 2 × 150-bp paired-end sequencing per flow cell with a mean of 120 million reads (range: 8 to 398 million) per sample.

A BD Rhapsody Single-Cell Analysis System (BD Biosciences) was used for the scRNA-seq analysis. In brief, PBMCs were processed into a single-cell suspension and loaded into a BD Rhapsody cartridge with >200,000 microwells. Then, a bead library was loaded into the microwell cartridge to saturation, and each cell was paired with a microbead. Next, the bead-cell complexes were hybridized with mRNA molecules to capture the barcoded oligos on the beads after lysing the cells in the microwell cartridge. The beads were collected into a single tube to generate a multiplex PCR-based library customized by the BD Rhapsody Immune Response Targeted Panel for Human (BD Biosciences). Fastq files were processed by an Illumina HiSeq 3000 platform and then processed into the expression matrix Fastq by the BD Rhapsody Analysis Pipeline. BD DataView software (BD Biosciences) and the R package Seurat (V 4.01) were used to analyze the expression matrix. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed using the R package “enrichplot” (Yu et al., 2012 (link)). The raw expression data from these experiments are available at the NCBI Gene Expression Omnibus database, with the following identifier: GSE175429 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE175429).

Healthy plant tissue (100 mg) and severely diseased plant tissue (100 mg) were homogenized in liquid nitrogen for DNA extraction for Illumina sequencing to serve as controls for MinION sequencing. DNA of healthy boxwood was extracted using Invisorb Spin Plant Mini Kit, and DNA of severely diseased boxwood was extracted using ZymoBIOMICS DNA Miniprep Kit.
Whole-genome sequencing of healthy boxwood was performed on an Illumina Nova Seq 6000 Platform (2 × 150 bp) at Novogene Corporation Inc. (Sacramento, CA). Low-quality reads and adapters were removed by the company. Illumina sequencing of severely diseased plant tissue was performed on an Illumina HiSeq 3000 Platform (2 × 100 bp) at the Iowa State University DNA Facility using six out of 96 barcodes (thus using 6/96th of a single run), and the quality of reads was checked using FastQC v0.11.9²⁷ . Reads were trimmed using Trimmomatic v0.39^{28 (link)} to remove adapters.

The NEC cells at 75 days, EC, and SE collected from different Petri dishes of Jin668 were pooled (each type have pooled six samples) and immediately frozen in liquid nitrogen (Figure 1d). Genomic DNA was extracted from NEC, EC, SE and leaves using the Plant Genomic DNA kit (TIANGEN, Cat. #DP305‐03). As well, the leaves of Y668 (WT) and the leaves of R0, R2, R4 regenerated offspring plants were extracted Genomic DNA. First, 2 μg of high‐quality genomic DNA was fragmented between 300 and 500 bp by sonication. Then, Illumina adapters were ligated following the manufacturer's protocols, after which fragments with adapters were treated with bisulphite using the EZ DNA Methylation‐Gold™ Kit (Catalog No. D5005). At the same time, unmethylated lambda DNA (Promega) was treated as a control. Finally, the treated DNA fragments were amplified and cleaned up with AMPure Beads. The paired‐end sequencing of the BS‐Seq library was performed on the Illumina HiSeq 3000 platform.

Fresh fecal contents were directly collected from the rat's cecum at the end of the study and stored in Sample Protector (TaKaRa, Dalian, China) at −80°C. The MoBio Power Fecal DNA Isolation kit (Mo BioLaboratories, Carlsbad, CA, USA) was used for DNA extraction. The quality of the extracted DNA was examined by agarose gel electrophoresis, and the OD 260/280 was analyzed by spectrophotometry. DNA libraries were prepared from 2 μg of total DNA for each sample using TruSeq DNA LT Sample Prep Kit v2 (Illumina, San Diego, California). Metagenomic sequencing was performed on HiSeq 3000 platform (Illumina, San Diego, California). After removing adapters, the raw reads were filtered to remove low-quality reads and reads that belong to the host. These high-quality reads from the samples were then assembled to contigs using Meta-Velevt. MetaGeneMark was employed to predict open reading frames (ORFs). In addition, a metagenomic catalog was generated based on the samples obtained in this study. Furthermore, the high-quality clean paired-end reads from each sample were aligned by BWA version 0.5.7-6 to the reference genes. Then the relative abundance of genes was predicted as described previously [15 (link)].

Liver tissues from three healthy adult Yorkshire pigs at Iowa State University were grounded into powder in liquid nitrogen using pestle and mortar. Total RNA was extracted using the Animal Tissue RNA Purification Kit (Norgen Bioteck Corp., Thorold, ON, Canada) per the manufacturer’s instructions. The total RNA from each sample was used for stranded RNA-seq library construction separately by using the Quantseq 3′ RNA-Seq Library Prep Kit FWD for Illumina (Lexogen GmbH, Vienna, Austria). Indexed libraries for individual samples were pooled together equimolarly and sequenced using an Illumina Hiseq3000 platform to generate 50 base single end reads from ends distal to poly(A)/poly(T) ends.