RNA-Seq of Biofloc Shing gut : Biofloc Shing gut 2 sample
Total genomic DNA from each samples was extracted using the phenol-chloroform method. DNA concentration and purity were measured on 1% agarose gel. According to the concentration, DNA was diluted to l ug/L using sterile water. 16S rRNA/18S rRNA/ITS genes of distinct regions 16S V3-V4 were amplified using specific primer 515F-806R with the barcode (515 F GTGCCAGCMGCCGCGGTAA and 806 R GGACTACHVGGGTWTCTAAT). All PCR reactions were carried out 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.Amplicon metagenomics analysis was conducted at the Novogene Company (China). Sequencing libraries were generated using TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, USA) following the manufacturer's recommendations, and index codes were added. The library quality was assessed on the Qubit@2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an Illumina NovaSeq 6000 platform, and 300 bp paired-end reads were generated for V3-V4 variable regions of the 16S rRNA gene.