Community Sequencing of V4 Region of Bos taurus indicus Rumen Samples
Total rumen contents were homogenized. Phenol:chloroform and beadbeating extraction was used to extract total DNA. Using custom primers designed by Kozich et al (2013), PCR reactions were performed with 50ng DNA, 10umol primers, 12.5uL 2x KAPA HotStart ReadyMix, and water to 25uL total. Custom primers were designed to amplify the V4 region of the 16S rRNA gene, and included adapters suitable for sequencing on Illumina technology and indices to allow for mulitplexing. PCR products were gel extracted, quantified, and equimolarly pooled. Using a MiSeq v2 2x250, a 10pmol library was sequenced with a 10% PhiX control. Sequences were demultiplexed after sequencing using Illumina BaseSpace.