16S sequencing of rumen epithelium: 10MR-post weaning
Microbial DNA was isolated using the QIAamp cador Pathogen Mini Kit (QIAGEN, Hilden, Germany). Genomic DNA was used to PCR-amplify the V4 hypervariable region of 16S rRNA gene using 5 Prime HotMasterMix (5 Prime, Hamburg, Germany) with primers (GTGBCAGCMGCCGCGGTAA, GGACTACHVGGGTWTCTAAT) described by Kozich et al., 2013 (doi: 10.1128/AEM.01043-13) and Hugerth et al., 2014 (doi: 10.1128/AEM.01403-14). The PCR conditions were as follows: 95C for 2 min, 35 cycles of 95C for 30 s, 50C for 60 s and 72C for 90 s and a final extension for 10 min at 72C. Duplicates of PCR products were pooled and purified using a SequalPrep Normalisation Plate (Thermo Fisher Scientific). The DNA libraries were parallel sequenced for 2 x 250bp paired-end reads using the HiSeq PE Rapid Cluster Kit v2 and HiSeq Rapid SBS Kit v2 on the rapid-run mode of the HiSeq 2500 system (Illumina). Sequence data were de-multiplexed and converted into FASTQ files using the bcl2fastq2 conversion tool.