Low TMAO Recipient Mouse 2
Stool samples from human donors were stored as fecal slurries (0.15g/ml) in 10% glycerol at 80C. Gnotobiotic C57BL/6 mice (aged 810 weeks) were maintained in sterile isolators for the duration of the experiment. Mice were colonized with 200l of a 5 dilution of the above stock by oral gavage after being transferred to a chemically defined diet supplemented with 1% w/w choline (Envigo TD.09041). 5 days following colonization, Rose Bengal Cold Light stroke model was performed. 24 hours post stroke injury, tissues were collected and immediately frozen at 80C. The 16S rRNA V4 region was amplified and sequenced using protocols mentioned in the Earth Microbiome Project (EMP). Raw 16S amplicon sequence and metadata were demultiplexed using split_libraries_fastq.py script implemented in QIIME1.9.1. The demultiplexed fastq file was split into sample specific fastq files using split_sequence_file_on_sample_ids.py script from Qiime1.9.1. Individual fastq files without non-biological nucleotides were processed using Divisive Amplicon Denoising Algorithm (DADA) pipeline.