16S amplicons of G. portentosa gut microbiome
DNA was purified using NucleoSpin soil DNA isolation kit (Macherey-Nagel, Dren, Germany) with SL1 lysis buffer and enhancer SX as described by the manufacturer. A cell disruptor (FastPrep-24, MP Biomedicals, Irvine, USA) was used for 30 s 2 at 5 m/s speed to improve lysis. PCR was performed using primers with Illumina adapters and targeting the 16S rRNA gene hypervariable regions V3-V4 (underlined) Klindworth MiSeq-B-V3-F (5-3). TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG) and MiSeq-B-V4-R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC [25] and Phusion high fidelity DNA polymerase (New England Biolabs, Ipswich, USA). PCR program involved heating at 98 C for 1 min, followed by 25 cycles of 98 C for 45 s, 60 C for 45 s and 72 C for 30 s, and a final extension step at 72 C for 5 min. Each sample was amplified in 50 L final volume reaction in triplicate and successful amplification was confirmed by 1% agarose gel electrophoresis (size ~460 bp). The PCR products from the same sample were combined and purified using the GeneRead Size Selection kit (QIAGEN, Germantown, USA) following manufacturer instructions, eluting with 30 L EB buffer. The sequencing was performed in a Illumina MiSeq, 2 x 300 paired-reads