16S rRNA fecal microbiome mouse Fluoxetine
For Illumina sequencing, Genomic DNA was amplified using the Earth Microbiome Project barcoded primer set, by adding nine extra bases in the adapter region of the forward amplification primer that support paired-end sequencing. The V4 region of the 16S rRNA gene (515F-806R) was amplified with region-specific primers that included the Illumina flowcell adapter sequences.  The reverse amplification primer also contained a twelve base barcode sequence that supports pooling of up to 2,167 different samples in each lane. Each 25 ul PCR reaction contains 12 ul of MoBio PCR Water (Certified DNA-Free), 10 ul of 5 Prime HotMasterMix (1x), 1 ul of Forward Primer (5 uM concentration, 200 pM final), 1 ul Golay Barcode Tagged Reverse Primer (5 uM concentration, 200 pM final), and 1 ul of template DNA. The conditions for PCR are also follows: 94C for 3 minutes to denature the DNA, with 35 cycles at 94C for 45 s, 50C for 60 s, and 72C for 90 s; with a final extension of 10 min at 72C to ensure complete amplification. The PCR amplicons were quantified using PicoGreen (Invitrogen) and a plate reader. Once quantified, different volumes of each of the products are pooled into a single tube so that each amplicon is represented equally. This pool was then cleaned up using UltraClean PCR Clean-Up Kit (MoBIO), and then quantified using the 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.