Cloning of cDNAs encoding A. rubens neuropeptide precursors

Total RNA from radial nerve cords dissected from adult A. rubens was isolated using the SV Total RNA Isolation System (Promega, Southampton, UK) and then used to synthesize cDNA using the QuantiTect® Reverse Transcription Kit (QIAgen, Manchester, UK). The cDNAs encoding neuropeptide precursors were amplified by PCR using Phusion® or Q5® High-Fidelity DNA Polymerase (NEB, Hitchin, Hertfordshire, UK) and the specific primers listed in Table ​Table1.1. PCR cycling conditions used varied depending on the primers and size of the amplicon. The size of the amplicon was determined using gel electrophoresis. PCR products were either gel extracted or column purified using the QIAquick® Gel Extraction Kit (QIAgen, Manchester, UK) before being blunt-end cloned into either pBluescript SKII (+; Agilent Technologies, Stockport, Cheshire, UK) or pCR®-Blunt II-TOPO® vector (Thermo Fisher Scientific, Paisley, UK). Plasmids with the correct sized amplicon were then isolated and sequenced (Eurofins Genomics GmbH, Ebersberg, Germany).

Sequences of primers used to clone cDNAs encoding A. rubens neuropeptide precursors.