Pippinht

Post-purification, libraries were individually size-selected using a Pippin HT (Sage Science) and 2% w/v agarose gel cassettes (Sage Science). The Pippin HT was set to collect products in the range of 240 to 360 bp. Proper size selection was validated by running a 1 µL aliquot on a BioAnalyzer (Agilent) using a High Sensitivity DNA kit (Agilent, cat. # 5067–4625).

EV microRNA was isolated using the miRNeasy kit (QIAGEN, Germantown, MD) and the concentrations of RNA was assessed on Bioanalyzer with an RNA (Pico) chip (Agilent Technologies, Santa Clara, CA). An in-house small RNA sequencing library construction method was used to characterize the microRNA in samples (PMID: 29388143). Proper insert size of the library was selected with Pippin HT (Sage Science, Beverly, MA) and the concentration was measured using NEBNext Library Quant Kit (New England Biolabs, Ipswich, MA). The library concentrations were adjusted and pooled to a final concentration of 2 nM then run on a NEXTseq DNA sequencer (Illumina, San Diego CA). The small RNA (sRNA-Seq) data was analyzed with sRNAnalyzer (PMC5716150). The quantity of individual microRNAs was determined based on the number of mapped reads that were adjusted with Count Per Mapped Million (CPM). EV mRNA was characterized using Agilent 8x60 microarray and fluorescent probes were prepared from isolated RNAs using Agilent QuickAmp Labeling Kit according to the manufacturer’s instructions (Santa Clara, CA). Gene expression information was obtained using Agilent’s Feature Extraction and processed with the Institute for Systems Biology’s in-house SLIMarray pipeline (PMC1636632).

High‐molecular‐weight DNA was extracted from a single pooled sample of insects (0.5 mL, ~50 individuals) using a previously established chloroform:isoamyl phase separation protocol (Jaworski et al., 2020 (link)). HMW DNA was size checked by Femto Pulse System (Agilent), and 10 μg of DNA was sheared to appropriate size range (15–20 kb) using Megaruptor 3 (Diagenode). The sheared DNA was concentrated by bead purification using PB Beads (PacBio). The sequencing library was constructed following the manufacturer's protocols using SMRTbell Express Template Prep Kit 2.0. The final library was size selected on a Pippin HT (Sage Science) using S1 marker with a 10–25 kb size selection. The recovered final library was quantified with Qubit HS kit (Invitrogen) and sized on Femto Pulse System (Agilent). The sequencing library was sequenced with PacBio Sequel II Sequencing kit 2.0, loaded to one 8 M SMRT cell, and sequenced in CCS mode for 30 h. RNA was extracted from similar pooled‐samples (N = 3) using the ZYMO (Irvine, CA, USA) Direct‐zol RNA miniprep kit (Cat. # R2050) and sequenced using NovaSeq (Illumina, San Diego, CA, USA) paired‐end (150 bp) sequencing performed by Novogene (Sacramento, CA, USA). RNA libraries were prepared by Novogene following their standard mRNA‐seq services (polyA capture followed by cDNA reverse transcription).

Aliquots (containing 2.24 ng DNA) of the same DNA samples from the four O. australiensis genotypes described above were prepared for Illumina short read sequencing. Short-read libraries were created using the Illumina Nextera tagment DNA enzyme (TDE1) according to Jones et al.^{28 (link)}. Libraries were size selected for 350–600 bp fragments using a PippinHT (Sage Science). Sequencing was performed on an Illumina NovaSeq 6000 S4 flow cell 300 cycles (150 bp paired end), being multiplexed with other projects. Sequencing was performed at the Biomolecular Resource Facility, ANU.

Total RNA was extracted from human liver tissue using MagMAX kit (ThermoFisher). Strand-specific RNA-seq libraries were prepared from 1 µg RNA using KAPA stranded mRNA-Seq Kit (KAPA Biosystems). Twelve-cycle PCR was performed to amplify libraries. The amplified libraries were size-selected at 400~600 bp using PippinHT (Sage Science). Sequencing was performed on Illumina HiSeq^®2500 (Illumina) by multiplexed paired-read run with 2X100 cycles.