rRNA sequence of chicken cecal content:adult female chicken
The V4 region of the 16S rRNA gene (515F-806R) was PCR amplified with region-specific primers that included sequencer adapter sequences used in the Illumina MiSeq flowcell. Briefly, sample DNA was amplified in a 25 l PCR reaction system contains 9.5 l of MO BIO PCR Water (Certified DNA-Free), 12.5 l of QuantaBios AccuStart II PCR ToughMix (2x concentration, 1x final), 1 l each Golay barcode tagged Forward Primer and Reverse Primer (5 M concentration, 200 pM final), and 1 l of template DNA. Amplicons are then quantified using PicoGreen (Invitrogen) and a plate reader (Infinite 200 PRO, Tecan). Once quantified, volumes of each of the products are equally pooled into a single tube. The pool was cleaned up using AMPure XP Beads (Beckman Coulter), and then quantified using a fluorometer (Qubit, Invitrogen). After quantification, the molarity of the pool is determined and diluted down to 2 nM, denatured, and then diluted to a final concentration of 6.75 pM with a 10% PhiX spike for sequencing on the Illumina MiSeq.