Multi-modal single cell analysis reveals gut microbiota reshapes brain immune landscape in aged mouse model.

We used 16S V3/V4 region amplification to evaluate the composition of bacteria species in mouse fecal pellets. Fecel pellets were collected from young-adult (12 weeks old) wild type C57Bl/6 mice and aged (72 weeks old) wild type C57Bl/6 mice after 21 days of vehicle or antibiotics treatment (to induce gut microbiota depletion). In one sequencing round, we sequenced a total of 12 different fecal samples (3 young control, 3 aged control, 3 young depleted gut microbiota (ABX) and 3 aged depleted gut microbiota (ABX)). Amplicons were indexed using the Nextera XT Index Kit and pooled into a library for Illumina sequencing. Fecal pellets were obtained from six 12 week old female C57Bl/6 mice and six 72 week old female C57Bl/6 mice . Three young-adult and three aged mice received vehicle treatment and had an unperturbed gut microbiota and three young-adult and three aged mice received antibiotics treatment and had a depleted gut microbiota. All 12 fecal pellet samples (3 from each group) were individually processed to extract DNA using the ZymoBIOMICS DNA Miniprep Kit. Following DNA extraction, the V3/V4 regions of the 16S rRNA gene were amplified. Amplicons were indexed using the Nextera XT Index Kit. The libraries were normalized and multiplexed into a single pool prior to validation by Qubit High-Sensitivity dsDNA, Agilent Bioanalyzer DNA 7500 Chip, and Kapa Illumina Library Quantification qPCR analyses. The final library (8.5pM) and PhiX (0.7pM) were sequenced on the MiSeq System using MiSeq V2 500 cycle kit (250-PE with 8-bp dual indexing). Base calling and demultiplexing were performed by MiSeq Controller Software.