Euwallacea fornicatus

Illumina RNA Sequencing of 10x pooled Euwallacea fornicatus male whole bodies

The poly(A) mRNA isolation was performed using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) or Ribo-Zero rRNA removal Kit (illumina). The mRNA fragmentation and priming was performed using NEBNext First Strand Synthesis Reaction Buffer and NEBNext Random Primers. First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase and the second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix. The purified double-stranded cDNAby AxyPrep Mag PCR Clean-up (Axygen)was then treated with End Prep Enzyme Mix to repair both ends and add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using AxyPrep Mag PCR Clean-up (Axygen), and fragments of ~360 bp (with the approximate insert size of 300 bp) were recovered. Each sample was then amplified by PCR for 11 cycles using P5 and P7 primers, with both primers carrying sequences which can anneal with flow cell to perform bridge PCR and P7 primer carrying a six-base index allowing for multiplexing. The PCR products were cleaned up using AxyPrep Mag PCR Clean-up (Axygen), validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). Then libraries with different indices were multiplexed and loaded on an Illumina HiSeq instrument according to manufacturers instructions (Illumina, San Diego, CA, USA). Sequencing was carried out using a 2x150bp paired-end (PE) configuration; image analysis and base calling were conducted by the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument.

{'sex': 'male', 'host': 'Acer negundo', 'tissue': 'Whole body, adult', 'isolate': '10x pooled', 'dev_stage': 'Adult', 'collected_by': 'DPIRD WA', 'geo_loc_name': 'Australia: Western Australia, Perth', 'identified_by': 'DPIRD WA', 'biosamplemodel': 'Invertebrate', 'collection_date': '2022-10-10', 'env_broad_scale': 'Suburban', 'isolation_source': 'Whole body insect', 'specimen_voucher': 'ANIC CSIRO', 'host_tissue_sampled': 'Whole body'}

Oxford Nanopore PromethION RNA sequencing of 8x pooled Euwallacea female whole bodies

Libraries prepared for long-read sequencing following the ONT PCR-cDNA Barcoding Kit SQK-PCB111.24 by the BRF-JCMSR ANU. Libraries were sequenced on an ONT PromethIon across two FLO-PRO002 R9.4.1 flow-cells. Basecalling of the fast5 reads to fastq was performed with the Guppy basecaller (RRID:SCR_023196) v7.1.4.

{'sex': 'not determined', 'host': 'Acer negundo', 'tissue': 'Whole body, adult', 'isolate': '8x pooled', 'dev_stage': 'Adult', 'collected_by': 'DPIRD WA', 'geo_loc_name': 'Australia: Western Australia, Perth', 'identified_by': 'DPIRD WA', 'biosamplemodel': 'Invertebrate', 'collection_date': '2022-10-10', 'env_broad_scale': 'Suburban', 'isolation_source': 'Whole body insect', 'specimen_voucher': 'ANIC CSIRO', 'host_tissue_sampled': 'Whole body'}

RNAseq of Euwallacea fornicatus: whole individual (female) adult females

polyA libraries were prepared with KAPA mRNA HyperPrep Kit (Roche) and sequenced with PE100bp reads on a Illumina Novaseq 6000

{'age': 'adult', 'sex': 'female', 'tissue': 'whole individual', 'isolate': '30', 'collected_by': 'Edeli Reyes, ereyesg@ucr.edu', 'geo_loc_name': 'USA:California', 'biosamplemodel': 'Invertebrate', 'collection_date': '2022-04-18', 'biomaterial_provider': 'Richard Stouthamer, richards@ucr.edu'}