What is the experimental workflow for Nuclear Yeast library construction and screening?

1、Library Construction:
Total RNA extraction → mRNA isolation & purification → Double-stranded cDNA synthesis & purification → In vitro ligation of ds cDNA into pGADT7 vector → E. coli library culture → Library plasmids → Transformation into Y187 yeast host → Y187 yeast library culture.

2、Two-Hybrid Screening:
(1) Mating method:
Bait recombinant pGBKT7 plasmid construction → Autoactivation test: Co-transformation of bait recombinant plasmid & empty pGADT7 into Y2HGold yeast host → Mixing Y2HGold yeast culture containing bait with Y187 yeast library culture → Plating on selective plates based on autoactivation results → Picking positive colonies → Culturing positive yeast & plasmid extraction → PCR identification of positives → Sending for sequencing → Sequencing result analysis.
(2) Co-transformation method:
Bait recombinant pGBKT7 plasmid construction → Autoactivation test: Co-transformation of bait recombinant plasmid & empty pGADT7 into Y2HGold yeast host → Transformation of library plasmids into Y2HGold competent cells containing bait → Plating on selective plates based on autoactivation results → Picking positive colonies → Culturing positive yeast & plasmid extraction → PCR identification of positives → Sending for sequencing → Sequencing result analysis.

3、One-Hybrid Screening:
Bait recombinant pABAi plasmid construction → Autoactivation test: Transformation of linearized bait recombinant plasmid & empty pGADT7 into Y1HGold yeast host → Transformation of library plasmids into Y1HGold competent cells containing bait → Plating on selective plates based on autoactivation results → Picking positive colonies → Culturing positive yeast & plasmid extraction → PCR identification of positives → Sending for sequencing → Sequencing result analysis.