16S amplicon sequencing of Mus musculus gut microbiome
DNA was extracted using the FastDNA Spin Kit for Soil (MP BiomedicalsTM) following the manufacturers instructions. A 2-step PCR-approach was used to amplify the V1-V2 variable region of the 16S rRNA gene. PCR with primers 27Fbif and 338R containing part of the sequencing primer sites as short overhangs (given in italics) (ACGACGCTCTTCCGATCTAGRGTTHGATYMTGGCTCAG and GACGTGTGCTCTTCCGATCTTGCTGCCTCCCGTAGGAGT, respectively) was used to enrich for target sequences (20 cycles). A second amplification step of 10 cycles added the two indices and Illumina adapters to amplicons according to PMID. Obtained products were pooled in equimolar ratios and sequenced on an Illumina MiSeq (2X300 bases, San Diego, USA).
812G