16S rRNA gene sequencing of rat intestinal microbiome:  Colon lumen after sham surgery
DNA was amplified by PCR using the specific 16S rRNA gene V4 region primer pair 515F/806R. These primers had a barcode region on the forward primer specific to the sample. PCR was performed using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94C for 3 minutes, followed by 28 cycles of 94C for 30 seconds, 53C for 40 seconds and 72C for 1 minute, after which a final elongation step at 72C for 5 minutes was performed. In total 30 PCR cycles (5 cycles used on PCR products were done. PCR products from all samples were pooled in equal proportions based on their molecular weight and DNA concentrations followed by purification using calibrated Ampure XP beads. A DNA library from the pooled products was prepared by following the Illumina TruSeq DNA library preparation protocol