16S rRNA of mus musculus: fecal matter
Genomic DNA was isolated from fecal matter using the QIAamp PowerFecal Pro DNA Kit (Qiagen, Hilden, Germany) according to the manufacturers instructions. The quality and quantity of DNA samples were estimated using a Qubit 2.0 fluorometer (Thermo Scientific, MA, USA). The amplicon-PCR (Polymerase chain reaction) was used to amplify the V3-V4 regions of the 16S rRNA gene by using 341F (5'- CCTACGGGNGGCWGCAG-3') and 805R (5'-GACTACHVGGGTATCTAATCC-3'). The amplified PCR product was purified and tagged with Illumina index adapters using the Nextera XT index kit (Illumina CA, USA). KAPA HiFi HotStart ReadyMix (2X) (Roche, Basel, Switzerland) was used for all PCR reactions. Any left-over short DNA fragments were removed by performing the clean-up PCR2. The purified libraries were then subjected to quality check on Qubit 2.0 fluorometer (Thermo Fisher Scientific, MA, USA) and finally, the integrity and size of the DNA were analyzed using Agilent 2100 Bioanalyzer system (Agilent, Singapore) using DNA chips (Agilent Technologies, Waldbronn, Germany). The prepared library was pooled and loaded to MiSeq with 251x2 paired-end sequencing.