Mite collection
In March 2020, Varroa mites were collected from managed hives in Onna village, Okinawa by removing honey bees from 2 frames onto a tray with icing sugar, and shaking the tray to remove mites from honey bees (Fig. 3). From the site of collection to the laboratory, mites were in icing sugar. Once at the laboratory, we discarded dead mites. Mites were divided into the following treatment preservation conditions: 1) snap-frozen and stored at -80 °C 2), immersed in absolute ethanol at room temperature, 3) immersed in absolute ethanol at 4 °C, 4) immersed in TRIzol (Thermo Fisher Scientific, Tokyo, Japan) at room temperature, and 5) immersed in RNAlater (Thermo Fisher Scientific, Tokyo, Japan) at room temperature. When immersed in a solution, 500 µL was used and each treatment consisted of 6 mites placed in separate tubes. Each treatment group was subjected to DNA and RNA extractions at intervals of 5, 10, 15, and 21 days for subsequent DNA and RNA quantity and quality evaluations. Snap-freezing and storage at -80 °C were chosen for the control group as it is a widely used method for specimen preservation [6, 15, 16].
Mite preparation for extractions
Mites were removed from the solution in which they were stored to new 1.5-mL tubes (Eppendorf catalog #0020125215) and chilled in liquid nitrogen. A clean, autoclaved pestle (Sigma Aldrich catalog #Z359947) was also chilled in liquid nitrogen to grind each mite in its tube. By visual inspection, ground mite powder that remained on the pestle was used for DNA extraction, ensuring that about half of the mite homogenates remain in the tubes for RNA extraction (Fig. 3).
DNA extraction
Mite DNA was extracted using a QIAamp DNA Micro kit (Qiagen, Tokyo, Japan), according to the manufacturer’s protocol. DNA quantity was determined using a Qubit fluorometer (Thermo Fisher Scientific, Tokyo, Japan), and quality was evaluated by the A260 / A280 ratio using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Tokyo, Japan). Eluted DNA was stored at -20 °C until further applications.
RNA extraction
Mite RNA was extracted using TRIzol according to the manufacturer’s protocol. Due to the small amount of mite present, the protocol was modified by using 50% of specified reagent volumes. Total RNA quality and quantity were evaluated using absorbance ratios of A230/260 and A230/280 on a Nanodrop spectrophotometer.
Library preparation
DNA library preparation
DNA libraries were prepared using a Nextera XT DNA Library Prep kit (Illumina, Tokyo, Japan) according to the manufacturer’s protocol, with optimization for mite DNA, using reagents at 20% of their specified volume. DNA was visualized by running electrophoresis on 1% agarose gels, for 20 min at 135 V.
RNA library Preparationp
RNA libraries were prepared using an NEBNext Single Cell/ Low-Input RNA Library kit (New England BioLabs, Tokyo, Japan), according to the manufacturer’s protocol. Purified cDNA was indexed with i5 and i7 primers (catalog #7600S, New England BioLabs, Tokyo, Japan), and then purified and size selected for a range of 400 to 2000 bp using 11% and 11.5% PEG and DynaBeads [19].
Both DNA and RNA libraries, as well as total RNA, were analyzed with a Bioanalyzer 2100 (Agilent, Tokyo, Japan) or a 4200 Tapestation (Agilent, Tokyo, Japan). For Bioanalyzer, high-sensitivity DNA kits and RNA pico kits (Agilent, Tokyo, Japan) were used, while for the Tapestation, a high-sensitivity D5000 kit (Agilent, Tokyo, Japan) was used.
Sequencing and analysis
A MiSeq (Illumina, Tokyo, Japan) was used to perform both DNA and RNA sequencing, at 50 cycles and read with single read-only. We were interested in validating the protocol and estimating mapping percentages, so longer read lengths or higher coverage were not necessary. Raw sequence data from the MiSeq were first analyzed with FastQC [20] for quick quality control to see if adapter removal was necessary and to ensure that sequenced data was of sufficient quality. DNA sequence data were then analyzed using Bowtie2 [21], Samtools [22], and Picard tools [23] to map to the reference genome (Vdes_3.0 [5]) and to identify duplicates. RNA sequence data were analyzed using Bowtie2 and Samtools, and then taxonomically classified using Kraken [14]. We compared this protocol with previously published RNA-seq libraries deposited on NCBI, which were mapped to the reference genome in a similar way (SRR8867385 [24], SRR5109825 & SRR5109827 [8], SRR533974 [25], SRR5760830 & SRR5760851 [26], SRR8100122 & SRR8100123, SRR5377267 & SRR5337268, and SRR8864012 [27]).