Uncoated 384 well plate

Cell lysates were diluted 1 : 2 in nuclease-free water. An Agilent Bravo was used to add 3 µl of lysate to individual wells of an uncoated 384-well plate (Corning). An NEB Luna Universal Probe One-Step RT-qPCR kit was used to carry out qPCR reactions. A single master mix containing enzyme mix, buffer and nuclease free water was split into aliquots on a round-bottomed 96-well plate (Nunclon), to which Taqman Gene Expression Assays (ThermoFisher) were manually added. The Bravo was then used to transfer 23.5 µl of reaction mix to the wells with lysate and to mix the lysates and reaction mix, and then transfer 4.5 µl from each mini-master mix to four wells in a 384-well rtPCR plate. Reactions were carried out on a Roche Lightcycler 480 with conditions as follows: one cycle at 55°C for 10 min and 95°C for 1 min, followed by 50 cycles at 95°C for 10 s and 60°C for 30 s.

Cell lysates were diluted 1 : 2 in nuclease-free water. An Agilent Bravo was used to add 3 µl of lysate to individual wells of an uncoated 384-well plate (Corning). The exact amount of starting RNA was not calculated as we were not comparing between test and control experiments. qPCR reactions were performed using an NEB Luna Universal Probe One-Step RT-qPCR kit, following the manufacturer's directions. A single master mix containing enzyme mix, buffer and nuclease free water was split into aliquots on a round-bottomed 96-well plate (Nunclon). Taqman Gene Expression Assays (ThermoFisher) were manually added. A 23.5 µl aliquot of the reaction mix was transferred to the lysate using the Agilent Bravo and mixed. From each well, 4.5 µl was transferred to four wells in a 384-well rtPCR plate. Reactions were carried out on a Roche Lightcycler 480 with conditions as follows: one reverse transcriptase incubation at 55°C for 10 min and 95°C for 1 min, followed by 50 cycles at 95°C for 10 s and 60°C for 30 s.