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Here, we show how to design and perform BFG-PCA experiments.

== This is still under construction ==

== The protocol may be deposited elsewhere ==

Table of contents

(Steps where multiple approaches are possible based on preference is shown as a,b,..)

1. Designing the space

   • Controls
   • Target space

2. Preparing the barcoded expression vectors

   • Available resources
   • Preparing pENTRY clones
   • Preparing barcoded destination vectors
     1. Random barcoding of destination vectors
     2. RCP-PCR
   • Strategies to create barcoded expression vectors
     1. One-by-one LR cloning
     2. En masse LR followed by identification
     3. In Yeast Assembly based barcoding

3. Estimate the required experiment scale

   • Monte-Carlo Simulation

4. BFG-PCA screening

   • Yeast strain preparation
   • Haploid cell culture
   • En masse mating
   • Diploid selection
   • Selection
   • Dox induction
   • Plasmid prep
   • Library PCR
   • Sequencing run
   • Data analysis
     • Barcode calling
     • Normalization
     • Performance measure
     • Visualization