Establishment of a humanized microbiome for analyzing the role of the microbiome in mediating the beneficial effects of ketogenic diet in colorectal cancer and its analysis by 16S gene sequencing. null

In this study, the beneficial effect of ketogenic diet in the context of colorectal cancer was analyzed using a CRC mouse model with a humanized gut microbiome. For this purpose, single homogenized human stool samples from five different healthy donors were thawed and suspended in a sterile 20% glycerol PBS solution at 10mg of stool per mL (5HD mix), aliquoted and frozen at -80°C for further use. To investigate how closely the 5HD mix resembled the original donor samples, the mix and the original human samples were analyzed by 16S amplicon sequencing. Briefly, DNA was extracted using the DNeasy PowerSoil DNA Isolation Kit (Qiagen), according to the kit manual, except that samples were homogenized using the TissueLyzerII for five minutes at 40Hz instead of intense vortexing followed by determination of concentrations and purities of the extracted DNA samples using a Nanodrop 2000 spectrophotometer purification and concentration using the DNA Clean and Concentrator Kit (Zymo Research). Prepared libraries were quantified using the Qubit® dsDNA BR Assay Kit (Thermofisher Scientific) and Qubit® 3 Fluorometer (ThermoFisher Scientific) and sample quality was assessed using the Bioanalyzer High Sensitivity DNA Analysis Kit (Agilent Technologies). Finally, 5 ng of DNA extracted was subjected to amplicon sequencing of the V4 region of 16S rRNA genes using primers 515F and 805R on a MiSeq instrument (Illumina) using 2×300 base pair read lengths.