title: v4 16S of Colobus vellerosus
design: We amplified the bacterial v4 region of the 16S ribosomal RNA gene using the following 515F and 806R primers containing 5 Illumina adapter tails and dual indexing barcodes: 515F 5 AATGATACGGCGACCACCGAGATCTACACTAGATCGCTATGGTAATTGTGTGCCAGCMGCCGCGGTAA; 806R 5 CAAGCAGAAGACGGCATACGAGATTCACCTAGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT. We set the PCRs with 12.5 ml NEB Q5 Hot start 2x Master mix, 1.25 ml 10uM Primer mix, 1 ml template DNA, and 10.25 ml MoBio certified DNA free water and used the following cycling protocol: 98 degrees for 30 seconds (1x) followed by 98 degrees for 10 seconds, 61 degrees for 20 seconds, and 72 degrees for 20 seconds (20x), followed by 72 degrees for 2 minutes and 4 degrees. The amplification products were cleaned up using Ampure XP beads and normalized into a final pool with an Eppendorf liquid handling robot. Libraries were sequenced as part of a 150bp paired-end sequencing run on the Illumina NextSeq platform following the manufacturers protocol.