16S sequencing of jejunal digesta: pair feeding
Microbial DNA was isolated using the DNeasy PowerLyzer PowerSoil kit (QIAGEN, Hilden, Germany). Genomic DNA was used to PCR-amplify the V4 hypervariable region of 16S rRNA gene using GoTaq G2 Hot Start polymerase (Promega, Mannheim, 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), 10 min at 72C. Duplicates of PCR products were pooled and purified using 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 software, v2.19 (Illumina).