Tguide s96 magnetic soil dna kit

The rhizosphere soil attached to the soil was collected by eluting with phosphoric acid buffer solution [38 (link)] as follows: The roots were placed in 25 mL of phosphate buffer solution (6.33 g NaH₂PO₄·H₂O, 16.5 g Na₂HPO₄·7H₂O, 200 μL Silwet L-77 L⁻¹; pH 7.0) in a 50 mL sterile centrifuge tube and vortexed for 15 s at the maximum speed on a vortex oscillator (Vortex-Geneie 2, Kezhida Information Technology Co., Ltd., Beijing, China). The suspension obtained by oscillation was filtered through a 100 μm sterilized nylon mesh filter into a new centrifuge tube and centrifuged at 3200× g for 15 min. The supernatant was removed, and the precipitate was collected as the rhizosphere soil and stored at −80 °C. The total DNA was extracted from the rhizosphere soil sample with the TGuide S96 Magnetic Soil DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China). The DNA concentration was measured using the Qubit dsDNA HS Assay Kit on a Qubit 4.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions.

Genomic DNA was extracted from 0.3 g sediment samples with the TGuide S96 Magnetic Soil DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China). The quality and integrity of extracted DNA was assessed with the Thermo Scientific Microplate Reader (Multiskan GO, America). The primer sets targeting 16S rRNA genes of microbes (Zhang and Lu, 2016 (link); Chen et al., 2021 (link)) and functional genes associated with microbial carbon metabolism (Zheng et al., 2018 (link)) were selected for PCR amplification as described in Supplementary Tables 2, 3. 16S rRNA sequencing was performed on the Illumina NovaSeq 6000 platform, and HT-qPCR QMEC was used for genetic quantification (Zheng et al., 2018 (link)). Both of projects were carried at Meige Technology Co., Ltd., Guangzhou, China. Bioinformatic analysis of amplicon sequencing and detailed protocols of HT-qPCR QMEC were performed as described in S1. The sequences reported in this study have been deposited in NCBI SRA database with the BioProject numbers PRJNA681135 and PRJNA674461 (Wang B. L. et al., 2021 (link)). Shannon index and niche breadth of microbes and carbon-functional genes were conducted with the “vegan_2.6-2” and “spaa_0.2.2” package, respectively.

Total genomic DNA was extracted from 27 samples using the TGuide S96 Magnetic Soil DNA Kit (Tiangen Biotech (Beijing) Co., Ltd.) according to manufacturer’s instructions. DNA was detected on a 1% agarose gel, and the concentration was determined using a spectrophotometer NanoDrop 2000 (NanoDrop Technologies, United States). The hypervariable region V3–V4 of the bacterial 16S rRNA gene were amplified with primer pairs 338F: 5′-ACTCCTACGGGAGGCAGCA-3′ and 806R: 5′-GGACTACHVGGGTWTCTAAT-3′. Both the forward and reverse 16S primers were tailed with sample-specific Illumina index sequences to allow for deep sequencing. The PCR was performed in a total reaction volume of 10 μL: DNA template 5–50 ng, forward primer (10 μM) 0.3 μL, reverse primer (10 μM) 0.3 μL, KOD FX Neo Buffer 5 μL, dNTP (2 mM each) 2 μL, KOD FX Neo 0.2 μL, and finally ddH2O up to 20 μL. After with initial denaturation at 95°C for 5 min, followed by 20 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 40 s, and a final step at 72°C for 7 min. The amplified products were purified with Omega DNA purification kit (Omega Inc., Norcross, GA, United States) and quantified using Qsep-400 (BiOptic, Inc., New Taipei City, Taiwan, ROC). The amplicon library was paired-end sequenced (2 × 250) on an Illumina novaseq6000 (Beijing Biomarker Technologies Co., Ltd., Beijing, China).