Mantis microfluidic dispensing system

10 technical replicates of 3 biological replicates of polyA+ RNA were plated in a 384 well PCR plate (Applied Biosystems) using a Mantis microfluidic dispensing system (FORMULATRIX), facilitating precise dispensing at small volumes. RNA was prepared by mixing 15 ng of polyA-enriched RNA in two conditions: One containing 10 units of MazF enzyme (Takara), MazF buffer (40mM Na2P04 pH 7.5, 0.05% Tween 20), and 1 U of RNaseOUT; and the other containing MazF buffer and RNaseOUT only. Cleavage and control reactions were incubated at 37 °C for 30 min and then heat denatured for 4 min to stop the cleavage reaction. MazF treated and control samples where reverse transcribed using the High-Capacity RNA-to-cDNA (Thermo Fisher) kit according to the manufacturer’s protocol at 1/4 volume added using Mantis microfluidic dispensing system. Differential MazF cleavage of the target RNA transcript was quantified by qPCR with IQ Multiplex Powermix (Bio-Rad) using two TaqMan probes (Bio-Rad) one targeting the cleavage site the other targeting an uncleaved section of the target transcript. All reactions were performed and quantified on a CFX384 Real-Time PCR Detection System (Bio-Rad).