Table 2.

Overview of options to consider in B3H experimental design.

Choice	Options	Recommendations
Reporter Strain (details in Table 3)	hfq+ vs. Δhfq	Δhfq behaves best for Hfq and ProQ interactions and is recommended for new interactions if Hfq may compete.
	F’ episome marked with tetR vs. kanR	Choose based on other antibiotic needs in system (e.g. F’ providing tetracycline resistance is well suited for use with deletion alleles from Keio collection)30
pAdapter plasmids (details in Table 2)	IPTG-inducible (pKB989) vs. constitutive expression of DNA-RNA Adapter (pCW17 or p35u4)	pCW17 recommended for ProQ-RNA interactions; p35u4 for Hfq-sRNA interactions
Transformation Style	Direct Inoculation from Bulk Transformation	Ideal for 96-well transformations for qualitative readout; able to screen a large number of constructs quickly
	Inoculation from Single Colonies	Ideal for quantitative readout; recommended for 48 or fewer transformations; provides access to biological replicates from individual colonies from a single transformation
Readout	Qualitative vs. Quantitative	These work well to perform in tandem and have different sensitivities. Since one readout may be ideal for a certain interaction, it is recommended to try both.
Counter assay for Mutagenesis Screen	No counter assay	You could choose to sequence all hits from your primary screen. If screening for mutants with a loss-of-interaction from a random mutagenesis library, it is anticipated that many hits will be undesired mutations such as premature stop codons.
	Secondary Interaction	If a protein-protein B2H interaction is available that would be orthogonal to the RNA interaction being disrupted, this is a straightforward way to ensure functionality of mutants isolated in the primary screen using the same detection strategy. Counter-screening with another RNA bait in a B3H setup can identify mutants with RNA-specific binding defects.
	Dot Blot (Immunodetection)	Only reports on expression levels of a mutant, not functionality; requires low endogenous expression relative to the prey fusion protein and an antibody specific to the prey.