2 wk post heavy wet-up soil 16S rRNA gene + P replicate C
DNA from soil using the Powersoil Pro kit following manufacturers protocol. The hypervariable region v4 of the 16S rRNA gene was amplified using primers 515FY (515F-GTGYCAGCMGCCGCGGTAA) and 806R (806R-GGACTACNVGGGTWTCTAAT). For 16S rRNA gene amplification, 0.1 L-1 of 1:20 diluted DNA was mixed with 1X Kapa HIFI Hotstart Readymix and 200 nM primers. The amplification protocol included an initial denaturation step at 95 C for 3 min, followed by 25 cycles of denaturation at 95 C for 30 s, annealing at 55 C for 30 s, extension at 72 C for 30 s, and a final extension step at 72 C for 5 min. After PCR, cleanup was performed using 0.8X AMPure XP Beads. Subsequently, a secondary amplification was carried out using Kapa HIFI mastermix with Illumina XT indexes to attach barcodes to the PCR products. A final cleanup step was performed using 1.2X AMPure XP Beads, followed by pooling of samples in equimolar ratios The resulting amplicons libraries were then sequenced on the Illumina MiSeq platform using a 2x250 cycle kit with approximately 25% phiX spike-in.