microbial 16s sequencing: of homo sapiens
Human fecal microbiota DNA was isolated by using ZymoBIOMICS 96 DNA Kit (ZymoBIOMICS) according to the manufacturers instructions. The variable region 4 of the 16S rRNA genes was amplified using primers: Forward 5- TATGGTAATTGTGTGCCAGCMGCCGCGGTAA -3 Reverse 5- AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT -3. PCR products were quantified using Quant-IT dsDNA high sensitivity assay (Invitrogen) according to the manufacturer's instructions.  Gel electrophoresis was used to check the success of PCR amplification. The concentration of the PCR product was measured by the Quan-IT dsDNA high sensitivity assay.  ~120ng of DNA of each PCR product was pooled together to generate an aggregated library for downstream processing. PCR DNA amplicons between 300-500bp were selected from the aggregated library on a targeted size selection platform (pippin prep 1.5% agarose cassette from Sage Sciences) according to the manufacturers instructions. The size of DNA amplicons was characterized on an Agilent Technologies 2100 bioanalyzer trace.
4-Pre-SG