Scyphophorus_acupunctatus_Mich3
The V3-V4 region of the bacterial 16S rRNA gene was amplified from gDNA derived from gut samples using the 16S rRNA Forward Primer = 5 TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG and the 16S rRNA Reverse Primer = 5 GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C, to generate an amplicon of ~550 bp (Klindworth et al., 2013). The 25 L PCR reactions contained 12.5 L of DreamTaq Green PCR Master Mix (2X; Thermo Scientific), 125 nM each of forward and reverse 16S rRNA gene primers, and 2 L (65140 ng) of gDNA with a thermal cycling program as follows: initial denaturation for 3 min at 95 C, followed by 30 cycles of denaturation for 15 s at 95 C, annealing for 30 s at 53 C, extension for 60 s at 72 C, and a final extension for 7 min at 72 C. PCR products were resolved on a 1.8% agarose gel. PCR for the no-template control was included. The libraries were prepared following the instructions of the 16S Metagenomic Sequencing Library Preparation for Illumina. The concentration of libraries was assessed by a Qubit 2.0 Fluorometer (Thermo, USA). Amplicon clean-up was carried out using AMPure XP beads (Beckman Coulter). Amplicons were indexed with the Illumina sequencing adapters using the Nextera XT Index Kit, purified, and quantified as previously described. The library's quality was assessed on the Agilent Bioanalyzer 2100 system. Indexed amplicons were sequenced in a paired-end (2300 bp) sequencing format with a MiSeq Reagent Kit V3 (600 cycles) using the MiSeq platform (Illumina, San Diego, CA, USA). Sequencing was performed in the Sequencing Unit at the Instituto de Ecologa, A.C. (Inecol), Mxico.