Metagenomic sequencing MRSA sample 2 of lung macrophages from methicillin-treated C57BL/7N mice infected with MRSA
Fecal specimens were collected from mice infected with SA or MRSA and treated with PBS, triptolide, triptolide combined with SM-164, or methicillin. Samples were collected in sterile tubes, immediately frozen, and stored at 80C until further analysis. Fecal DNA was extracted using a HiPure Stool DNA Kit (Cat # D314102, Magen Biotechnology Co., Ltd). Genomic DNA was randomly fragmented by Covaris to an average size of 300-350 bp. The fragments were treated with End Prep Enzyme Mix for end repairing, 5 Phosphorylation and 3 adenylated, to add adaptors to both ends. Each sample was then amplified by PCR for 8 cycles using P5 and P7 primers, with both primers carrying sequences which can anneal with flowcell to perform bridge PCR and P7 primer carrying a six-base index allowing for multiplexing. The PCR products were cleaned up and validated using an Agilent 2100 Bioanalyzer. The qualified libraries were sequenced with paired-end 150-bp reads on the Illumina NovaSeq 6000 System. The quality statistics software cutadapt (v1.9.1) was used to remove primers and low quality sequences. The BWA software (v0.7.12) was used to compare with the host genome and filter out the reads that might be of host origin. The remaining sequences were aligned to the NR database using diamond (v2.1.10.164) for taxonomic annotation. The relative abundance of taxa at different taxonomic levels (phylum to species) was calculated by summing the abundances of annotated genes in each sample. Microbiome diversity analyses were performed using the R package vegan (v2.6.4). Alpha diversity analysis was performed using the Simpson index. Differential taxa and functional features between groups were identified using Metastats method.