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. 2022 Feb 14;18(2):e10629. doi: 10.15252/msb.202110629

Figure 1. The MaMTH‐HTS workflow.

Figure 1

1) Grow up cell lines containing stably integrated prey and reporter. 2) Transfect cells with Bait of Interest. 3) Induce the cells with tetracycline to express Bait and Prey. Continue to grow the cells for 48 h. At this point, we will have a mixed population containing the cells expressing combinations of Bait (along with tagBFP/blue fluorescence) and Prey (along with mCherry/red fluorescence). The cells expressing interacting Bait/Prey pairs will induce eGFP reporter and have additional green fluorescence. 4) Carry out sequential FACS sort, first selecting the cells with red fluorescence (indicating proper Prey expression), then blue fluorescence (indicating proper Bait expression) and finally green fluorescence (indicating Bait/Prey interaction). 5) Expand/grow the cells to increase overall cell number, isolate genomic DNA and PCR amplify using prey cassette specific primers. 6) Deep sequence amplified PCR products to identify prey ORFs enriched in population (indicating potential interactors).