16S rRNA sequencing raw data of Microcystis aeruginosa and cyanotoxin in grass carp intestine
16S rRNA sequencing raw data of Microcystis aeruginosa and cyanotoxin in grass carp intestine
Grass carp (Ctenopharyngodon idella) were fasted for 24 h prior to sampling to minimize intestinal content interference. Following anesthesia with MS-222, fish were euthanized humanely, and the body surface was disinfected with 70% ethanol. Under aseptic conditions, the entire intestine was carefully dissected using sterile instruments. Intestinal contents or mucosal scrapings were collected with sterile forceps and spatulas, immediately transferred into pre-labeled sterile cryogenic tubes, and snap-frozen in liquid nitrogen. Samples were subsequently stored at −80°C until DNA extraction. All tools were sterilized between samples to prevent cross-contamination, and detailed metadata including sample ID, treatment group, and sampling date were recorded. Approximately 150–200 mg of frozen intestinal content or mucosal scraping was used per sample. DNA was extracted using a commercial microbial DNA kit (e.g., Qiagen DNeasy PowerSoil/PowerFecal Pro) following the manufacturer's protocol with a bead-beating step (2 × 30 s at high speed) to ensure efficient cell lysis. After mechanical disruption, samples were subjected to chemical lysis and inhibitor removal, and DNA was bound to the spin column, washed, and eluted in 50 µL nuclease-free water. A negative extraction control was processed in parallel. Extracted DNA was quantified by Qubit, purity checked by A260/280, and integrity verified by agarose gel electrophoresis; DNA was stored at −20°C (short term) or −80°C (long term) until 16S library preparation. The 16S rRNA V3–V4 region was amplified using primers 341F/806R with a high-fidelity polymerase. PCR products were purified with AMPure XP beads, indexed in a second PCR, and quantified using Qubit. After quality assessment and equimolar pooling, libraries were sequenced on the Illumina MiSeq platform (2×300 bp) with 10–20% PhiX control. Negative extraction and PCR controls were included throughout the process.