Roboinject system

Plasmids were linearized using NotI before in vitro synthesis of 5’ capped cRNA using the T3 mMessage mMachine transcription kit according to manufacturer's protocol (Thermo Fischer Scientific). The cRNA was purified using the GeneJET RNA purification kit (Thermo Fischer Scientific). Xenopus oocytes were placed individually into 96-well plates and injected with 50 nL of a total 500 ng/uL RNA using the Roboinject system (Multi Channel Systems GmbH). Injected oocytes were incubated at 16° C in ND96 until the day of recording, typically between 1-2 days post injection. The GPCR-containing plasmids were co-injected with plasmids containing different mouse G-protein inward rectifying potassium channels (GIRKs) in order to record changes in current upon GPCR activation. This was done in a 1:2 ratio (GIRK:GPCR). For evaluation of G-protein identity, 50 pg of pertussis toxin (PTX) was injected into previously injected oocytes, 6h prior to recording.

cRNA was synthesized in vitro using the T3 mMessage mMachine transcription kit according to manufacturer protocol to include a 5′ cap (Thermo Fisher Scientific). Before injection RNA was purified using the GeneJET RNA purification kit (Thermo Fisher Scientific). Size-sorted and defolliculated Xenopus oocytes (Ecocyte) were placed individually into 96-well plates and injected with 50 nl of 500 ng/µl RNA using the Roboinject system (Multi Channel Systems). When two constructs were coinjected, the total RNA concentration remained at 500 ng/µl, with a 1:1 ratio of the components. Injected oocytes were incubated at 16°C in ND96 solution until the day of recording, typically between 3 and 6 d postinjection.

5'-capped cRNA was synthesized in vitro using the T3 mMessage mMachine transcription kit according to manufacturer’s protocol (Thermo Fischer Scientific, CA, USA). RNA was then purified using the GeneJET RNA purification kit (Thermo Fischer Scientific, CA, USA) prior to cRNA injection. Defolliculated Xenopus oocytes were placed individually into 96 well plates and injected with 50nL of 500ng/μL RNA using the Roboinject system (Multi Channel Systems GmbH, Reutlingen, Germany). When two constructs were injected the total RNA concentration remained 500ng/μL, with a 1:1 ratio of the components. Injected oocytes were incubated at 16°C in ND96 until the day of recording, typically between 3-5 days post injection.