Novaseq6000 sequencing machine

Two fecal samples were collected from each cat using a fecal loop with and without lubrication (N = 14 total; Supplementary Table S1) (32 (link)). At least 200 mg feces were used for DNA extraction by Qiagen Allprep PowerFecal DNA/RNA kit (Qiagen, MD), following the protocols provided by the manufacturer. During DNA extraction, fecal samples were homogenized by the Qiagen PowerLyzer24 instrument (Qiagen, MD) to achieve homogeneous results. The extracted DNA concentrations were measured by the Qubit 3 Fluorometer (Invitrogen, CA), and A260/A280 absorption ratios were assessed using the NanoDrop One C Microvolume Spectrophotometer (Thermo Fisher Scientific, MA). DNA fragmentation was performed by M220 Focused-ultrasonicator (Covaris, MA) on 1.5–2 μg of DNA for each sample to achieve fragmented DNA of ~500 bp. NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, MA) was used to construct WGS metagenomic sequencing libraries using the fragmented DNA. Final library concentrations and size distributions were measured by LabChip GX Touch HT Nucleic Acid Analyzer (PerkinElmer, MA) before being sequenced on an Illumina NovaSeq6000 sequencing machine on the 150-bp paired-end mode at the Genomics Service Laboratory at the HudsonAlpha Institute for Biotechnology (Huntsville, AL).

DNA/RNA extractions were conducted using Zymo Quick-DNA/RNA Miniprep Plus Kit (Zymo Research, CA) according to the manufacturer’s instructions. DNA/RNA quantity was measured using the Qubit Fluorometer 3.0 (Thermo Fisher Scientific, MA) with the Qubit RNA BR Assay Kit. RNA samples with >100 ng yield were selected for RNA-seq library preparation. RNA sequencing libraries were constructed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, MA) and NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, MA). The final concentrations and size distribution of the libraries were checked using the Qubit and HT DNA NGS 3K Assay on a LabChip GX Touch HT machine (Perkin Elmer, MA) (Supplementary Table S2). Libraries were sent for sequencing on an Illumina NovaSeq 6000 sequencing machine to generate 150-bp paired-end reads.