Fastdna spin kit

Whole genome DNA was extracted from bulk soil, rhizosphere, rhizoplane, and endospheric samples by using Fast DNATM Spin Kit, specialized for extracting DNA from soil, following the manufacturer’s instructions manual (MP Biomedical, Santa Ana, CA, USA). Then all the DNA samples were subjected to gel electrophoresis and further purification, using Universal DNA Purification Kits according to the manufacturer’s instructions (Tiangen Biotech Co., Ltd., Beijing, China). DNA was quantified by using Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA) before being stored at −20 °C for further molecular analysis.

The soil samples were collected to extract DNA according to the FastDNA^TM SPIN Kit (MP Biomedicals, USA)for Soil extraction specification, and agarose gel electrophoresis was used to detect DNA quality. DNA concentration detector One drop 2000 (Thermo, USA) was used to determine the concentration of the DNA. The bacterial 16S rRNA sequencing fragment was V3-V4, and the regional universal primer was 338 F:5’-ACTCCTACGGGAGGCAGCAG-3’, 806 R: 5’-GGACTACHVGGGTWTCTAAT-3’,^{16 (link)} the fungal ITS sequencing fragment was ITS2, and the regional universal primer was fITS7: 5’-GTGARTCATCGAATCTTTG-3’, ITS4: 5’-TCCTCCGCTTATTGATATGC-3’,^{17 (link)} PCR reaction system and program were performed formed following a previous study.^{18 (link)} PCR products were purifed with Gel Extraction Kit (Vazyme, China) and pooled in equimolar concentrations. Paired-end sequencing (PE300) of bacterial and fungal amplicons were carried out on an Illumina MiSeq platform at Hangzhou Lianchuan Biotechnology Co., Ltd (Hangzhou, China).

Total genomic DNA was extracted using the Fast DNATM Spin Kit (MP Biomedicals, LLC, Santa Ana, CA, United States). The DNA purity and quantity were determined by the NanoDrop 2000 spectrophotometer (Thermo Fisher) and agarose gel. Using the genomic DNA as a template, the hypervariable V3-V4 regions of the 16S rRNA gene were amplified by PCR with the primers 341F (5′-CCTACGGGNBGCASCAG-3′) and 785R (5′-CCTACGGGNBGCASCAG-3′) (Fadeev et al., 2021 (link)). The fungal ITS1 regions of ITS were amplified by PCR with the primers ITS5-1737F (5′- GGAAGTAAAAGTCGTAACAAGG-3′) and ITS2-2043R (5′- GCTGCGTTCTTCATCGATGC-3′) (Huang et al., 2016 (link)). The PCR amplification products were collected from a 2% agarose gel and purified by Vazyme VAHTSTM DNA Clean Beads. The sequencing libraries were established using TruSeq Nano DNA LT Library Prep Kit (Illumina, SD, USA) and then sequenced on an Illumina MiSeq platform (Biomarker Technologies Corporation, Beijing, China).

Base on the manufacturer’s instructions, Total DNA was extracted from an aliquot of 0.25 g of sediment from each sample using an MP Biomedicals Fast DNATM SPIN Kit. The extracted genomic DNA was detected by 1% agarose gel electrophoresis, and the purity and concentration were determined by a UV spectrophotometer (Eppendorf, Germany). The measured DNA sample was stored at − 20 °C for subsequent use.

DNA from tissue samples obtained from the larval gut, pupal gut, male and female moth gut, and tea leaves was extracted using the MP Biomedical FastDNA^TM Spin Kit following the manufacturer’s protocol. In the case of soil samples, a Qiagen DNeasy PowerSoil Kit (Hilden, Germany) was used for DNA extraction. The extracted DNA was then assessed for its quality using 0.8% agarose gel and was quantified using a Nanodrop-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The extracted DNA was subjected to amplification using specific primers and sequencing. In PCR, the adjusted DNA concentration of approximately 10 ng was used for fungi and bacteria. For bacteria, standard Illumina sequencing primers (515 FB and 806 RB) were used to amplify the V3-V4 region of the 16S rRNA gene [31 (link)]. While ITS3mix and ITS4ngs primers were used to target the ITS2 region of fungi [32 (link)]. The amplicons were then subjected to electrophoresis on 0.8% agarose gel and subsequently sequenced using Illumina MiSeq technology 300 bp (MiSeq Reagent Kit v3, San Diego, CA, USA) with the sequencing service provided by Sangon Biotech (Shanghai, China) Co., Ltd. China.

A FastDNATM Spin Kit (MP Biomedicals, Santa Ana, CA, USA) was used to extract DNA from 0.5 g of soil (fresh weight) following the manufacturer’s instructions. The extracted DNA was then purified using the PowerClean DNA Purification Kit (Mo Bio, Carlsbad, CA, USA), and its concentration and quality measured using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The fungal ITS1 and ITS2 regions were amplified using 20–50 ng of DNA as a template, using the upstream primer containing the GTGAATCATCGARTC sequence and the downstream primer containing the TCCTCCGCTTATTGAT sequence. The PCR reaction parameters across a total of 24 cycles were: pre-denaturation parameter 94 °C, 3 min; denaturation parameter 94 °C, 5 s; annealing parameter 57 °C, 90 s; extension parameter 72 °C, 10 s; final extension parameter 72 °C, 5 min. After the amplification products were detected, PE250 paired-end sequencing was performed with an Illumina MiSeq (Illumina, San Diego, CA, USA) instrument to obtain the raw sequencing data.

Bacterial genomic DNA extraction, amplification, and 16S rRNA gene sequencing of the metronidazole-treated SPF WT mice was carried out using a FastDNA^TM SPIN kit according to the manufacturer’s protocol (MP Biomedical, Aurora, CT, USA) by Chunlab Inc., Seoul, South Korea [44 (link)]. Briefly, the DNA was first amplified using barcoded primers specific to the V1 and V3 regions of the bacterial 16S rRNA genes. Sequences were then sorted out by their respective unique barcodes, and low-quality reads were removed. Bacterial 16S rRNA gene sequencing of 1 μg of PCR amplified product of each sample was done using the 454 GS FLX Titanium Sequencing System (Roche, Branford, FL, USA). Sequence reads were identified using the EZBioCloud Genome Database. Fecal microbial composition, diversity, and clustering were analyzed using the CLcommunity bioinformatics software provided by Chunlab Inc., Seoul, Korea.