1. Mouse Y1H pipeline.
Mouse Y1H pipeline. (A) Workflow to retrieve the mouse TF open‐reading frame (ORF) clone resource. We predict that the mouse genome contains 1576 TF‐coding genes (Materials and methods). We were able to retrieve cDNA templates for 1260 of these. To initiate the cloning of the corresponding ORFs, we first applied a strong selection scheme choosing the longest ORF per TF containing the respective DBD. After filtering, 1218 TF cDNA templates were retained. So far, 1014 TF ORFs have been cloned. The corresponding entry clones were sequence verified using high‐throughput sequencing. In all, 150 and 750 TFs were, respectively, partially or fully sequence verified. (B) The mouse Y1H screening pipeline using either the mating (diploid Y1H) or the direct transformation (haploid Y1H) protocol. The 745 TFs in Y1H‐compatible AD‐vectors are transformed into the yeast DNA bait strain or into the mating yeast strain for the transformation and the mating assays, respectively; the colonies are then selected and four replicates are created by using a colony‐pinning robot that transfers the 384 individually transformed yeast strains and spots them in a square pattern. In the mating protocol, the mating yeast strains containing the respective TFs are mated with the yeast DNA bait strain and then selected to create diploid yeast. Finally, in both protocols, TF–DNA bait interactions are identified based on growth on a selective 3‐amino‐1,2,4‐triazole (3‐AT)‐containing yeast plate. The green circles indicate positive protein–DNA interactions.
Carine Gubelmann et al. Mol Syst Biol 2013;9:682
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