Intestinal microbe of largemouth bass
Total genomic DNA was isolated from gut tissues using the TIANamp Stool DNA Kit according to the manufacturers instructions (TIANGEN BIOTECH (BEIJING) CO., LTD. China). Concentrations of extracted DNA were determined using a NanoDrop Spectrophotometer 2000c (NanoDrop Technologies, Wilmington, DE, USA). 16S rRNA genes of distinct regions (16S V3-V4) were amplified used specific primer (341F 5-CCTAYGGGRBGCASCAG-3 and 806R 5-GGACTACNNGGGTATCTAAT-3) with the barcode. All PCR reactions were carried out in 30 ?L reactions with 15 ?L of Phusion? High-Fidelity PCR Master Mix (New England Biolabs); 0.2 ?M of forward and reverse primers, and about 10 ng template DNA. Thermal cycling consisted of initial denaturation at 98for 1 min, followed by 30 cycles of denaturation at 98for 10 s, annealing at 50for 30 s, and elongation at 72 for 30 s. Finally 72 for 5 min. Mix same volume of 1loading buffer (contained SYB green) with PCR products and operate electrophoresis on 2% agarose gel for detection. PCR products was mixed in equidensity ratios. Then, mixture PCR products was purified with GeneJETTM Gel Extraction Kit (Thermo Scientific). Sequencing libraries were generated using Ion Plus Fragment Library Kit 48 rxns (Thermo Scientific) following manufacturer's recommendations. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific). At last, the library was sequenced on an Ion S5TM XL platform and 400 bp/624 bp single-end reads were generated.