title: Microbiota seq for rearing water
design: DNA was extracted from each pooled sample using Qiagens QIAamp DNA Kits according to instructions. The concentration of DNA was measured with a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, USA). The 16S rDNA V3 and V4 variable regions of the ribosomal RNA gene were amplified by using barcoded fusion primers  (341F 5-CCTAYGGGRBGCASCAG-3 and 806R 5-GGACTACNNGGGTATCTAAT-3). PCR reactions were conducted in triplicate for each sample as follows: initial denaturation at 95 for 5 minutes, followed by 30 cycles of denaturation at 95 for 1 minute, annealing at 60 for 1 minute, and elongation at 72 for 1 minute. The final elongation was carried out at 72 for 7 minutes. PCR products were extracted from 2% agarose gels, purified using the QIAquick Gel Extraction Kit from Qiagen, and quantified using the ABI StepOnePlus Real-Time PCR System from Life Technologies, USA. Finally, the clean-up DNA was pooled in equimolar and paired-end (PE250) sequenced using an Illumina HiSeq 2500 sequencing platform (Illumina, USA) at Gene Denovo Biotechnology Co. (Guangzhou, China) following standard protocols.