16S rRNA gene Amplicon Sequencing of mouse microbiota: mucin-amendment biological replicate MucC
DNA extracted from the mouse colon microcosms using a phenol-chloroform bead-beating protocol or obtained by whole genome amplification (WGA) of sorted cells was used as a template for PCR. PCR amplification was performed with a two-step barcoding approach. In the first-step PCR, the following oligonucleotide primers targeting the 16S rRNA gene of most bacteria were used as forward and reverse primers, respectively: S-D-bact-0341-b-S-17 (5-CCTACGGGNGGCWGCAG-3) and S-D-bact-0785-a-A-21 (5-GACTACHVGGGTATCTAATCC-3). These first step oligonucleotide primers contained head adaptors (5-GCTATGCGCGAGCTGC-3) in order to be barcoded in a second step PCR. Barcode primers consisted of the 16 bp head sequence and a sample-specific 8 bp barcode from a previously published list at the 5 end. The barcoded amplicons were purified with ZR-96 DNA Clean-up Kit (Zymo Research, USA) and quantified using the Quant-iT PicoGreen dsDNA Assay (Invitrogen, USA). An equimolar library was constructed by pooling samples, and the resulting library was sent for sequencing on an Illumina MiSeq platform at Microsynth AG (Balgach, Switzerland).