Qubit spectrophotometer

Murine corneal epithelium and conjunctival tissue were collected from wild-type C57BL/6 J mice according to previously published methods^{20 (link)}. In brief, total RNA was extracted with Qiagen RNeasy Plus Micro RNA isolation kit (Qiagen, Germantown, MD, USA) following the manufacturer’s protocol. One sample equaled the tissue pooled from both eyes of each animal. cDNA was synthesized using Ready-To-Go First-Strand beads (GE Healthcare Life Sciences, Marlborough, MA, USA) and random hexamers (Life Technologies, Grand Island, New York, USA) with 1 µg total RNA as template. DNA concentration was measured with a Qubit spectrophotometer (Life Technologies). Digital polymerase chain reaction (PCR) was performed as previously described with a QuantStudio 3D Digital PCR system (Life Technologies) with Slc5a8 Taqman assay primer set Mm00520629_m1 (Applied Biosystems, Inc. [ABI], Foster City, CA) and normalized by concentration of cDNA^{33 (link)}.

RNA was extracted using the RNA extraction kit (Macherey-Nagel, Düren, Germany) according to manufacturers’ instructions. Quantification was performed using the Qubit spectrophotometer (Invitrogen, Paisley, United Kingdom). cDNA was synthesized from equal RNA amounts using the PrimeScript 1st strand cDNA synthesis kit (Takara, Shiga, Japan).

The plant materials that were sequenced in this study were collected from the National Orchid Conservation & Research Center of Shenzhen, and the specimens were deposited in the National Orchid Conservation Center herbarium (NOCC) (Table 1). All of the plant materials were collected with the permission of this institution, and all of the samples were identified by Prof. Zhong-Jian Liu. We complied with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna. Detailed information about localities and samples are given in Table 1 and Figure 1. Specifically, we collected 109 samples were from 24 wild populations of D. catenatum, and 10 samples were from 2 wild populations of D. huoshanense. We selected 3–5 individuals from each population and sampled their leaves. The leaves were dried in sealed plastic bags that were filled with silica gel until the DNA extraction was performed.
The total genomic DNA was extracted using a Plant Genomic DNA kit (Tiangen, Beijing, China) according to the manufacturer’s protocol. For all of the samples, the DNA was quantified using a Qubit spectrophotometer (Invitrogen, Carlsbad, CA, USA).

Phage DNA was extracted using the Favorprep (Favorgen, Taiwan, China) viral nucleic acid extraction kit according to the manufacturer’s instructions (available on http://www.favorgen.com/favorgen/serv_1/mem_t1/h_1/pdf/rna/FAVNK%20000-1%20001%20001_1%20001-2_1503.pdf accessed on 23 October 2020) A Qubit spectrophotometer (Invitrogen, Waltham, MA, USA) and a NanoDrop 2000 system (Thermofisher Scientific, Waltham, MA USA) were used to assess the quality and quantity of the extracted nucleic acid. To ascertain the type of nucleic acid in the phage genomes, DNase I and RNase I (Invitrogen, Waltham, MA, USA) digestions were performed using the manufacturer’s protocol and visualised on a 0.8% agarose gel (Sigma Aldrich, Burlington, MA, USA) stained with a SYBR Safe in-gel stain (Invitrogen, Waltham, MA, USA) and ran at 45 V for 1 h.

For ITS2 amplicon-based DNA sequencing, 0.25 grams of each sample was aseptically weighed and subjected to total DNA extraction in a biosafety cabinet using DNeasy PowerSoil Kit (Qiagen, Germany) and then resuspended utilizing sterile water (UltraPure Distillated Water, Invitrogen, Carlsbad, California, USA). Contamination between samples was excluded using Ultrapure DNA free water as a separate blank control.
As requested by the sequencing service, samples and controls were checked for ITS2 DNA amplicon sequencing eligibility using specific tailed ITS3 and ITS4 primers for the ITS2 region. The DNA concentration required for metagenomics (range: 3–10 ng/μl) was determined by a Qubit spectrophotometer (Invitrogen, Carlsbad, California, USA).
ITS2 barcode sequencing and Sanger sequencing were performed using the NGS sequencing service offered by the BMR Genomics Company, Padua, Italy. Libraries were prepared using Nextera XT DNA Library with sequencing performed via the Illumina MiSeq Paired-End platform.
Sequences were uploaded on GenBank with the BioProject ID: PRJNA851016.

The V3-V4 regions within the 16S rRNA gene were amplified from the DNA extracts using the forward primer 341F (CCTACGGGNGGCWGCAG) and reverse primer 805R (GACTACHVGGGTATCTAATCC). Each primer was labeled with an Illumina adaptor sequence and a unique multiplex identifier code. The standard 50 μL polymerase chain reaction (PCR) system included 2x Phanta Max Master Mix (Vazyme, China), 10 μM forward and reverse primers, 5 μL of DNA template and 16 μL of ddH₂O. Thermal cycling program of 3 min initial denaturation at 95°C, followed by 8 cycles at 95°C for 30 s, 55°C for 30 s, 72°C for 45 s, and a final elongation step at 72°C for 5 min. The amplicons were quantified using Quant-It Pico Green kit (Invitrogen, United States) with Qubit Spectrophotometer (Invitrogen, United States), and samples were pooled together for library preparation. The library concentration was measured with Agilent 2100 Bioanalyzer (Agilent Technologies, United States), and diluted to 4 nM using Tris pH 8.5. After denaturation, 6 pM of the combined sample library and PhiX control was loaded on a MiSeq Platform (Illumina; United States) using 600 cycles MiSeq Reagent Kit PE300 v3 (Illumina; United States), and 300 bps paired-end reads were cluster generated.