Gut microbiota of Chinstrap penguins: senior
The primers 341F806R were used to amplify the V3V4 regions of the 16S rRNA genes in each sample, with Illumina adapters and unique barcodes added to the end of the reverse primer (Berg et al. 2012). All polymerase chain reactions (PCR) were carried out in 15 L of reaction mixture containing 7.5 L Phusion High-Fidelity PCR Master Mix (New England Biolabs), 1 L of dNTP (2.5 mM), 1 L of forward and reverse primers (10 M), and 1 L template DNA. Thermal cycling consisted of initial denaturation at 95C for 2 min, followed by 25 cycles of denaturation at 94C for 5 s, annealing at 55C for 30 s, and elongation at 72C for 30 s. The PCR products of each sample were detected by electrophoresis on 1.5% agarose gels, and the obtained PCR products were purified, quantified, and mixed in equal amounts to construct the sequencing libraries. The quality of the sequencing libraries was then determined using an Agilent Bioanalyzer 2100 system and Qubit@ 2.0 Fluorometer (Thermo Scientic, USA). Finally, the libraries were sequenced on an Illumina NovaSeq platform with 250 bp paired-end strategy.