We recommend the users of our protocols read our 2020 Nature Methods, Qin, X. et al. paper; Cell-type-specific signaling networks in heterocellular organoids before beginning. This will give an overview as to why we generated our TOBis barcodes instead of using the traditional Fluidigm barcoding method, the advantages of using TOBis over other barcoding methods and also applications of its use on intestinal organoids.

We have also published a very specific, step-by-step guide in carrying out the entire TOBis CyTOF protocol, as well as analysing PTM signalling data acquired using our specially developed CyGNAL package, in our 2021 Nature Protocols, Sufi, J. & Qin, X. et al. paper; Multiplexed single-cell analysis of organoid signaling networks.

Lastly, a guide of using the OT-2 software and writing protocols can all be found on their website.

I would like to acknowledge the aid of and thank:

• Dr Alaric Taylor, in helping me write the script and teaching me how to use the OT-2 machine.
• Ferran Cardoso (@FerranC96) for helping me set up the Tape Lab OT-2 GitHub repository. And for developing and maintaining the CyGNAL data analysis pipeline.
• And lastly, the TAPE Lab group for their continuous support in helping create the TOBis barcoding strategy.

The work here is actively being developed by Jahangir Sufi.