title: RNA-Seq of biofloc water 6
design: Sample processing:Biofloc samples (50 ml) were collected from triplicate aquaculture tanks per treatment (F: clear-water control; G: glucose; M: maltodextrin; GM: glucose-maltodextrin mix), homogenized, and centrifuged (10,000  g, 10 min). Pellets were stored at 80C.DNA extraction:Genomic DNA was isolated from 250 mg pellets using the DNeasy PowerSoil Pro Kit (QIAGEN, Cat# 47014) with bead-beating (45 sec, 6.0 m/s). DNA quality was verified by Nanodrop (A260/280 >1.8) and electrophoresis.16S rRNA amplification:The V3-V4 hypervariable region was amplified with:Forward primer: 5-CCTACGGGNGGCWGCAG-3 (341F)  Reverse primer: 5-GACTACHVGGGTATCTAATCC-3 (805R)  PCR conditions: 95C/3 min; 30 cycles of 95C/30s, 55C/30s, 72C/45s; final extension 72C/5 min.Library preparation:Amplicons were dual-indexed using Nextera XT Index Kit (Illumina) with 8-cycle PCR. Libraries were purified (AMPure XP beads), quantified (Qubit dsDNA HS Assay), and pooled equimolarly.Quality control:Library fragment size (~550 bp) was confirmed by Bioanalyzer (Agilent 2100). Pooled libraries were denatured with 0.2N NaOH and diluted to 6 pM.Sequencing:250-bp paired-end sequencing performed on Illumina MiSeq (San Diego, CA) using MiSeq Reagent Kit v3 (600-cycle).