DNA 16S amplicon sequencing Alouatta palliata palliata: fecal microbiome
DNA extractions were performed using a phenol-chloroform bead beating extraction protocol: we added 500 l phenol-chloroform:IAA to approximately 200 ul of fecal sample with an equal proportion of zirconium beads. The standard bead-beating method of extraction was modified by adding 20l of Proteinase K to each sample and digesting them for 2 hours at 56 C. After two rounds of successive bead beating and chilling on ice, the supernatant was then mixed with another aliquot of phenol-chloroform and centrifuged to leave a supernatant of solubilized DNA. This was left to precipitate overnight at -20 C with isopropanol and sodium acetate. The following day, chilled samples were centrifuged to pellet DNA and washed with ethanol. Then, washed DNA was solubilized in TE buffer, and purified using a spin column purification process as per the Invitrogen Quick PCR Purification Kit purification procedure. Extracted and purified DNA was then quantified using a Qubit 3 Fluorometer and frozen at -20 C until future use. We prepared 16S libraries for sequencing using a two-step amplification protocol. For PCR1, the primers Hyb515F_rRNA (5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGYCAGCMGCCGCGGTA -3) and Hyb806R_rRNA (5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT-3') to amplify a 292 base pair portion of the V4 region of the 16S gene.