V4 16S rRNA amplicon sequencing of 42 day old layer chicken: cecum
Cecal contents were collected after animal necropsy and immediately stored at 4 C. Cecal metagenomic DNA was isolated using the Qiagen MagAttract PowerMicrobiome DNA/RNA kit (Valencia, CA) following the manufacturers instructions. A Qiagen TissueLyzer was used for the bead beating step. Extracted DNA was used for the construction of a 16S rRNA gene library following a the wet-lab protocol from the Schloss lab. Briefly, Illumina indexed reads were created using PCR amplification of the V4 region of bacterial 16S rRNA gene. No PCR amplicons were observed in the negative control samples using water as the DNA template. Amplified DNA was normalized using the SequalPrep Normalization Plate (Invitrogen), and pooled into a single library for each 96-well plate.