title: 16S sequencing:_lobster 3 intestine healthy females
design: V3-V4 region of the bacterial 16S rRNA gene was amplified from the gut samples using the 16S rRNA Forward Primer = 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and the 16S rRNA Reverse Primer = 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC, 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 (65-140 ng) of gDNA. All runs were performed in a C1000 Thermal Cycler (Bio-Rad Laboratories Inc., USA) with a thermal cycling program as follows: initial denaturation for 3 min at 95C, followed by 30 cycles of denaturation for 15 s at 95C, annealing for 30 s at 53C, extension for 60 s at 72C, and a final extension for 7 min at 72C. PCR products were resolved on 1.8% agarose gel electrophoresis. PCR for the no-template control was included.