Table 2.

Troubleshooting guide for mitoribosome profiling and related protocols

Problem	Possible Cause	Solution
Low recovery of footprints	Low starting cell number	Start with more cells; some strains or mutants may have less mitochondrial translation.
	Low solubility	Increase buffer volume in cryogenic lysis
	Mitoribosome dissociation	Check lysis buffer conditions for ratio of monovalent to divalent cations. Do not vigorously shake (aerate) lysate.
	RNA degradation	Be sure all reagents are RNase-free and work area is free of dust; use barrier tips
Poor separation or smearing of RNA in gels	Incomplete ethanol/salt removal	Wash pellet thoroughly with vortexing in 70% ethanol; remove all trace of ethanol wash
	Incomplete resuspension of pellet	Mark position of pellet in tube before it dries
	Urea accumulation in gel wells prior to loading	Use a syringe to clear wells of urea within 2 minutes of loading
	RNA not fully denatured	Heat RNA in urea load buffer and place immediately on wet ice
High fraction of rRNA reads in mitoribosome library	Gel slice too big in initial size selection	Excise a more narrow range
	Mitoribosome dissociation	Check lysis buffer conditions for ratio of monovalent to divalent cations. Do not vigorously shake (aerate) lysate.
Poor signal on northern blot	Probe specific activity too low	Check labeling protocol and 32P stock
	Probe not full denatured	Boil probe for at least two minutes and place immediately on wet ice just before use
	High background	Repeat wash step with 2X or even 1X blot wash buffer
Poor/inconsistent fragmentation for RNA-seq	RNA is in too high a concentration of buffer	Tris concentration should be 1 mM before addition of 2X alkaline fragmentation buffer
	Condensation is forming during heating	Perform fragmentation in thermocycler with heated lid