3.0 fluorometer

Gut microbial genomic DNA was extracted from the cecum contents by using a TIANamp Stool DNA Kit (DP328, Tiangen), according to the manufacturer’s instruction. The V3-V4 region of the 16S rRNA gene was amplified using the well-established universal primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The amplification was performed as follows: Initial denaturation for 3 min at 95 °C, 30 cycles each of denaturation for 30 s at 95 °C, annealing for 30 s at 55 °C, and primer extension for 45 s at 72 °C. The amplicons were purified using the Ampure XP beads (A63881, Beckman Coulter, Brea, CA, USA) and quantified with a Qubit 3.0 fluorometer using a Qubit dsDNA HS Assay Kit (Q32854, Invitrogen, Carlsbad, CA, USA), before being sequenced on an Illumina Miseq PE300 platform by Hangzhou Kaitai Biotechnology Co.; Ltd. (Hangzhou, China).

To construct the library for PacBio sequencing, the qualified RNA from seven tissues, including the three testes and four ovaries, were mixed in equal amounts. The mixed RNA sample was reverse-transcribed for mRNA using the SMARTer™ PCR cDNA Synthesis Kit. PCR amplification was performed using the KAPA HiFi HotStart PCR Kit. Then, the PCR product for the SMRTbell library was constructed using the SMRTbell template pre kit. The concentration of the SMRTbell library was measured using a Qubit 3.0 fluorometer with a Qubit™ 1X dsDNA HS Assay kit (Invitrogen, Carlsbad, USA). The quantified criteria of library quality were concentration > 10 ng/μl with dispersive but continuous distribution in the range of 1–10kbp. A total of 2.5 ng of the library was sequenced for each SMRT cell using the binding kit 2.1 from the PacBio Sequel platform, producing 20 h of movies. The sample information was first registered as BioProject with accession number PRJNA532819 and BioSample with accession number SAMN11415730. Subsequently, the subread sequence generated by the PacBio Iso-Seq platform was deposited into the NCBI Sequence Read Archive (SRA) with accession number SRR7453063.

Permafrost sediments from three depths (3.4, 5.8, and 14.8 m) were used for simultaneous extraction of extracellular DNA (eDNA) and intracellular DNA (iDNA) using a modified protocol described in our earlier study [8 (link)]. Briefly, permafrost sediment (10 g) was mixed with a sterile phosphate buffer (0.12 M Na₂HPO₄ [pH 8]) and then centrifuged at 10,000×g for 10 min at 4 °C in order to separate the eDNA fraction into the aqueous phase. The remaining sediment was used to extract iDNA pool using DNeasy PowerMax soil kit (QIAGEN, Carlsbad, CA) according to the manufacturer’s procedures. The eDNA fraction in the supernatant was extracted using the standard procedures of the same kit except that the steps for bead beating and cell lysis were bypassed. A parallel extraction with sediment-free blank control was accompanied to monitor potential contamination introduced from the reagents and laboratory environment during extraction. The concentration of DNA was quantified using a Qubit 3.0 fluorometer with the dsDNA HS assay kit (Invitrogen, Carlsbad, CA, USA). Furthermore, the quality and size distribution of the iDNA and eDNA fractions were determined using Bioanalyzer DNA High Sensitivity chips (Agilent, CA). The DNA yield in the blank control was below detection (< 0.01 ng/μL) and was not included for metagenomic sequencing.

DNA was extracted from single female and male pupae using CTAB DNA extraction as described elsewhere [52 (link)]. Concentrations were measured on a Qubit 3.0 Fluorometer using the dsDNA BR Assay Kit (Invitrogen, Carlsbad, CA), and approximately 4 μg DNA per sample was used for DNA digestion reactions. DNA was double digested using NdeI × NotI, DraI × NheI (all Fermentas, Vilnius, Lithuania), or AgeI × BspHI (New England Biolabs, Ipswich, MA) enzymes (see S1 Methods for details). All digested DNA was separated by electrophoresis on a 1% TBE agarose gel and subsequently transferred by capillary transfer to an Amersham Hybond-N+ nylon membrane (GE Healthcare, Milwaukee, WI).
A probe specific to EkMasc with high homology to EkMascB (96%) was made by PCR-labeling (see S1 Methods for details). The probe was labeled with digoxigenin-11-dUTPs (Roche Diagnostics, Mannheim, Germany) using primers Masc_Sb_F and Masc_Sb_R. For Southern hybridization, 100 ng of the probe was used. The Southern blot assay was performed as described previously [55 (link)] with some modifications [56 (link)].

DNA extraction for the polymerase chain reactions employed a PowerSoil DNA Extraction Kit. The genetic material was quantified by fluorescence, using a Qubit 3.0 Fluorometer and a Qubit dsDNA BR Assay Kit (Invitrogen). After quantification, the extractions were diluted to a final total DNA concentration of 1000 ng/mL.

The quantity of cfDNA was determined with a Qubit 3.0 Fluorometer using a high-sensitivity Qubit dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA, USA).