A pool of approximately 400 fourth instars and adults aphids were individually opened and squeezed in 1 ml of PBS. After removing the aphids, each drop was transferred to 1.5 ml microfuge tube and gently centrifuged at 50g to pellet aphid cells and debris. The supernatant (containing symbionts) was then transferred to a new tube and spun at 8000g for 10 min at 4C to pellet symbionts. Total RNA was isolated from each strains using the mirVana miRNA Isolation Kit (Ambion, Thermo Fisher Scientific) followed by DNase treatment using the TURBO DNA-free Kit (Ambion, Thermo Fisher Scientific) and ethanol precipitation in the presence of glycogen. A total of three 400 aphids pool per strain were extracted generating 3 independent biological replicates. We sequentially depleted rRNA using the Ribo-Zero (Bacteria) Magnetic Kit (Epicentre, Illumina) according to manufacturers protocol followed by Ribo-Zero-Treated RNA purification using the Illumina modified RNeasy MinElut Cleanup Kit (Qiagen).  RNA templates were quality checked by the Georgia Genomics Facility using an Agilent 2100 Bioanalyzer (Agilent Technologies) and Fragment Analyzer (Advanced Analytical).  Standard, paired-end sequencing libraries (75 bp) were generated using the Kapa stranded RNA-seq Library Preparation Kit (Kapa Biosystems) and Illumina sequenced on a NextSeq (150 Cycles) Mid Output Flow Cell.

NextSeq 500 paired end sequencing; In-vivo RNAseq of symbionts from A. pisum ZA17 rep 3

BA_ZA17

We developed a centrifugation-based protocol in order to enrich H. defensa from the whole aphids to obtain a high quality in-vivo H. defensa transcriptome.

Candidatus Hamiltonella defensa in-vivo Transcriptome

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