16S rRNA sequencing of soil samples: tillage-reseeded alpine meadows in Tibet, June 2024
DNA was extracted from 0.5 g of sieved soil using the E.Z.N.A. Soil DNA Kit (Omega Bio-tek). The integrity of the extracted DNA was verified by 1.2% agarose gel electrophoresis, and its purity and concentration were determined using a NanoDrop 2000 spectrophotometer. The V3-V4 region of the 16S rRNA gene was amplified using primers 338F/806R (Klindworth et al., 2013) in a 25 L PCR reaction mixture. The reaction consisted of 5 FastPfu Buffer, 2.5 mM dNTPs, 10 M primers, and FastPfu Polymerase, with 10-20 ng of DNA template. PCR conditions were as follows: 98C for 5 min, followed by 25 cycles of 98C (30 s), 53C (30 s), and 72C (45 s), with a final extension at 72C for 5 min. The amplicons were purified, quantified using PicoGreen (Invitrogen), and pooled equimolarly. Sequencing was performed using paired-end (PE250) sequencing on an Illumina NovaSeq 6000 platform (Novogene, China).