Reagent	Volume	Final Concentration	Notes
10× Transcription buffer	2.0 µL	1×
2× NTP/cap mixture	6.0 µL	3.3×	The amount of NTP/cap mixture is altered from the manufacturer’s protocol to facilitate incorporation of fluorescent UTP
Cy3-UTP or Cy5-UTP	1.5 µL	0.375 mM	ChromaTide Alexa-Fluor UTPs (Thermo-Fisher) may also be used at a final concentration of 0.3 mM
Linearized DNA	(variable)	1 µg	Less than 1 µg can be used per reaction but will reduce yield
10× enzyme mix	2.0 µL	1×	Reactions using SP6 RNA polymerase require longer incubation times (4 hours) compared with those using T7 or T3 RNA polymerases (2 hours) for similar yields
Nuclease-free H2O	(variable)	-
Total	20 µl		Reaction may be scaled up for increased RNA production.