16S sequencing of Arboridia adanae: Apulia
The 16S rRNA gene V3-V4 region was amplified from the whole body in reactions of 25 l containing 1X the KAPA HiFi HS ReadyMix Buffer (Roche), and the primers 341F_ILL (5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3) and 805R-2_ILL (5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACNVGGGTWTCTAATCC-3)30,31 anchored with the Illumina forward and reverse overhang adapters to a final concentration of 0.3 M each and 100 ng of DNA (50 ng/l). PCR amplification controls (reactions with no DNA template) were included in each amplification process. The PCR conditions were 3 min at 95 C, 35x(30 s at 95 C, 30 s at 55 C, 90 min at 72 C), 7 min at 72 C, using a 96-Well Fast Thermal Cycler. Quality checks for amplification success and efficiency were performed by capillary electrophoresis using the QIAxcel Advanced System (QIAGEN). Bacterial amplicons were sequenced using Illumina MiSeq 2x300 bp with a minimum depth of 100,000 reads per sample.