Taqman qpcr probes

Subconfluent cultures were resuspended in TRIzol (ThermoFisher Scientific) and used immediately or otherwise stored at −70°C until use. RNA was isolated using Direct-zol RNA kit (Zymo Research). Once purified, RNA was transcribed to cDNA via High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). Resulting cDNA was then quantified utilizing Taqman Q-PCR probes in a QuantStudio 5 Real-Time PCR System (ThermoFisher Scientific). Data was then analyzed using Microsoft Excel and plotted in GraphPad PRISM.

Cell cultures were resuspended in TRIzol (Thermo Fisher Scientific; Waltham, MA, USA). RNA was isolated and purified (Direct-zol RNA kit; Zymo Research), reverse transcribed via High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), and quantified using TaqMan qPCR probes for TAS2R4, TAS2R8, TAS2R10, TAS2R13, TAS2R14, TAS2R39, TAS2R43, CASP3, CASP7, BAD, BAX, BCL-2, RyR1, RyR2, RyR3, IP3R1, IP3R2, IP3R3, FOXM1, GNAI1, GNAI2, GNAI3, GNAQ, GNAO1, GNAZ, GNAT3 and UBC (QuantStudio 5; Thermo Fisher Scientific). UBC was used as an endogenous control due to its stability in expression in cancer cells.^{154 (link)}

For gene expression experiments, cell harvesting and reverse transcription for cDNA generation was performed using a previously described modification^{27 (link)} of the commercial Cells-to-Ct kit (Thermo Fisher Scientific) 48 hours post-transfection. Transcript expression was then quantified with qPCR using Fast Advanced Master Mix (Thermo Fisher Scientific) and TaqMan qPCR probes (Thermo Fisher Scientific, Supplementary Table 7 and 8) with GAPDH control probes (Thermo Fisher Scientific). All qPCR reactions were performed in 5uL reactions with 4 technical replicates in 384-well format, and read out using a LightCycler 480 Instrument II (Roche). For multiplexed targeting reactions, readout of different targets was performed in separate wells.
Expression levels were calculated by subtracting housekeeping control (GAPDH) Ct values from target Ct values to normalize for total input, resulting in ΔCt levels. Relative transcript abundance was computed as 2^˄(-ΔCt). All replicates were performed as biological replicates
For analysis of RNA quality post-knockdown with LwaCas13a, total RNA was harvested by lysing cells using TRI Reagent^® and purifying the RNA using the Direct-zol RNA MiniPrep Plus kit (Zymo). 4 ng of total RNA was analyzed using a RNA 6000 Pico Bioanalyzer kit (Agilent).