title: AMPLICON sequencing of sediment metagenome
design: Co-extraction of DNA and RNA from 4 g of homogenized sediments or 1L seawater filters was carried out using the RNeasy Powersoil Total RNA Kit and RNeasy Powersoil DNA Elution Kit (QIAGEN, Germany). The extracted RNA was treated with TURBO DNA-free Kit (Invitrogen, USA) to remove DNA. Purified RNA was reverse transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany). PMA-treated DNA was extracted using the Powersoil Kit (QIAGEN, Germany). The V4 hypervariable region of the 16S rRNA gene was PCR amplified with the universal primers 515F (5-GTGYCAGCMGCCGCGGTAA-3) and 806R (5-GGACTACNVGGGTWTCTAAT-3) for bacteria. PCR reactions were conducted with an initial denaturation at 98 for 1 min, followed by 30 cycles of denaturation at 98 for 10 s, annealing at 50 for 30 s, and elongation at 72 for 30 s, with a final extension at 72 for 5 min. PCR products were purified with Universal DNA Purification Kit (TianGen, China), and sequencing libraries were generated using NEB Next Ultra II FS DNA PCR-Free Library Prep Kit (New England Biolabs, USA). Sequencing was performed on an Illumina Novaseq 6000 PE390 platform (Novogene, China).
identifiers.namespace: SUB14955498
identifiers.id: missing141