DNA extraction and amplicon sequencing
Soil DNA was extracted using the FastDNATM SPIN kit
(MP Biomedicals) according to the manufacturer’s protocol, after which
the concentration and quality were checked using a Nano-100, a NanoDrop
Spectro-photometer. Primers targeting the V3-V4 region of the16S rRNA
gene of bacteria, 338F (5´-ACTCCTACGGGAGGCAGCA-3´) and 806R
(5´-GGACTACHVGGGTWTCTAAT-3´). The PCR reaction consisted of 25 μl
2×Primer Taq (Takara, RR902A), 1 μl F-Primer (10 mM) and 1 μl R-Primer
(10 mM), 3 μl DNA and 20 μl dd-H2O. The PCR program was 94°C 5 min, 31
cycles of (94°C 30 s, 52°C 30 s, 72°C 45 s), 72°C 10 min. The ITS2
region of fungi, 3F (5´-GCATCGATGAAGAACGCAGC-3´) and 4R
(5´-TCCTCCGCTTATTGATATGC-3´), were chosen for amplification. The PCR
reaction consisted of 6 μl 10×E×Taq Buffer, 6 μl dNTP, 0.6 μl BSA, 0.3
μl E×Taq, 1 μl DNA, 1.2 μl F-Primer, 1.2 μl R-Primer, and 43.7 μl
dd-H2O. The PCR program was 94°C 5 min, 25 cycles of (94°C 30 s, 55°C 30
s, 72°C 30 s), 72°C 7 min. PCR was run on a Biorad 1000, and the ending
temperature was 16°C. PCR products was mixed in equimolar ratios
according to the GeneTools Analysis Software (Version 4.03.05.0,
SynGene). Next, the mix of PCR products was purified using an E.Z.N.A.
Gel Extraction Kit (Omega, USA). Sequencing libraries were generated
using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England
Biolabs, MA, USA) following manufacturer’s recommendations, and index
codes were added. The library quality was assessed on the Qubit@ 2.0
Fluorometer (Thermo Fisher Scientific, MA, USA). The library was
sequenced on an Illumina Nova6000 platform and 250 bp paired-end reads
were generated (Guangdong Magigene Biotechnology Co., Ltd. Guangzhou,
China).