Micro amp enduraplate optical 384 well clear reaction plates

Thermal unfolding of proteins was monitored in a 20 uL volume in Micro-Amp EnduraPlate Optical 384-well Clear Reaction Plates (ThermoFisher: cat no. 4483285 ). Reactions contained 50 mM HEPES pH 7.5, 62.5 mM NaCl, 7 μM 3CL^pro or PL^pro, 5% DMSO, and 4 × SYPRO-orange protein gel stain (ThermoFisher Scientific, Massachusetts, USA; cat no. S6651). Famotidine and the positive controls ML188 and compound 6 for 3CL^pro and PL^pro, respectively, were incubated with the protein for 15 min before the addition of SYPRO orange. Plates were covered with Micro-Amp Optical Adhesive Film (ThermoFisher Scientific, Massachusetts, USA; cat no. 4360954) and run on Applied Biosystems 7900HT (California, USA) real time PCR instrument. Samples were incubated at 25 °C for 2 min followed by an increase in temperature of 1 °C/min up to 95 °C. Fluorescence was monitored continuously. Each sample was run in triplicate and compounds were tested at 1 mM, 2.5 mM, and 5 mM. The melting temperature (T_m) was obtained from the first derivative of the raw thermal denaturing data were determined and smoothed to calculate melting temperature (T_m) values^{40 (link)}.

For qRT-PCR validation, RNA prepared from embryonic E15.5 forebrains, adult cerebral cortex and adult hippocampus was converted to complementary DNA (cDNA) using an Ambion RETROScript kit according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA). First, 100 ng of cDNA was used for the multiplex qRT-PCR reaction, combining a target gene and one of the two housekeeping genes Gapdh and Hprt. The target genes chosen for validation were Hspa13 [two copies in Ts1Cje and Ts65Dn mice and three copies in Dp(16)1/Yey mice], App [two copies in Ts1Cje mice and three copies in Ts65Dn and Dp(16)1/Yey mice], Ttc3 (three copies in all three models), and Rfx5 (two copies in all three models) (Table S29). Multiplex qRT-PCR was performed using TaqMan Multiplex Master Mix in MicroAmp™ EnduraPlate™ Optical 384-Well Clear Reaction Plates according to the manufacturer's instructions (Thermo Fisher Scientific). Amplification was conducted using a QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific). Data analysis was performed using the Expression Suite Software to determine the relative quantity of each transcript of interest (Thermo Fisher Scientific).