high fat diet+10% broccoli stalks (HFD+S) HS rep 1
The effects of each diet on the bacterial population in the gut microbiome was examined by analyzing the prokaryotic 16S ribosomal RNA gene (16S rRNA) which is approximately 1500 bp long and contains nine variable regions interspersed among conserved regions. These variable regions were subjected to phylogenetic classification, in diverse microbial populations. The following protocol describes a two-step PCR-based method for preparing samples for sequencing the variable V3 and V4 regions of the 16S rRNA gene. Bacterial DNA was extracted from fecal samples by using the DNeasy Powersoil kit (Qiagen) according to the manufacturers instructions. Each sample was then quantified with a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) and diluted to a final concentration of 5 ng L1 in 10mM Tris at pH 8.5. The 16S library preparation was carried out as described in Illuminas 16S sample preparation guide with minor modifications. PrimeStar HS DNA Polymerase Premix (Takara-Clontech, Mountain View, CA, USA) was used instead of the PCR enzyme.