title: 16S rrna of chicken: cecal sample
design: DNA extraction was performed using the Nucleospin kit (Macherey-Nagel, Germany). The DNA mass and purity were measured using the Nanodrop Spectrophotometer.Genomic DNA was diluted to a concentration of 10 ng/L per well in a volume of 10 L in a 96-well PCR plate and submitted to the Genomic Sciences Laboratory at North Carolina State University in Raleigh, NC, for library preparation. The V3-V4 hypervariable region of the 16S rRNA gene of the bacterial DNA was amplified using the following forward primer pairs: 16SF: 5' - TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT G GGN GGC WGC AG - 3' and 16SR: 5' - GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C - 3'. The PCR cycling conditions included an initial denaturation step at 95C for 3 minutes, followed by 25 cycles of denaturation at 95C for 30 seconds, annealing at 55C for 30 seconds, and extension at 72C for 30 seconds. A final extension step was performed at 72C for 5 minutes. Following the amplification cycles, the reaction mixture was held at 4C until further processing. After PCR amplification, the PCR products were purified using AMPure XP bead cleanup (Beckman Coulter Life Sciences). The DNA samples were quantified and normalized to a loading library concentration of 4 nM. The amplicons were subjected to paired-end sequencing on an Illumina MiSeq platform using the MiSeq V3 300x2 sequencing kit. The sequencing was performed with  25% PhiX control spikein.