GSM8490366: iRFP, 5 hr TNFα, rep1; Homo sapiens; OTHER
Cells were harvested for by centrifugation and lysed in TRI reagent (Sigma-Aldrich, T9424). To isolate RNA, 200 uL chloroform per 1 mL TRI reagent was added, shaken vigorously, incubated at RT for 5 min, then centrifuged at 12,000 g for 15 min at 4°C. The aqueous phase was transferred to a new tube, and 1 uL GlycoBlue (ThermoFisher, AM9515) and 500 uL 2-propanol per 1 mL TRI reagent was added. Samples were incubated at RT for 10 min then centrifuged at 12,000 g for 10 min at 4°C. The supernatant was removed from the RNA pellet, and the pellet was washed by adding at least 1 mL 75% ethanol. Samples were vortexed and centrifuged at 7500 g for 5 min at 4°C. The RNA pellet was briefly dried and then resuspended in water 400 ug of RNA was treated with Turbo DNase (ThermoFisher Scientific, AM2238) for 30 min at 37°C. 100 ug of RNA was reverse transcribed using Protoscript II and two gene-specific primers with UMIs at a 1:1 ratio. cDNA was treated with 1 L each of RNase A and RNase H (NEB M0297S and ThermoFisher, EN0531) for 30 min at 37°C. cDNA from each sample was purified by spin column then quantified by qPCR. cDNA was then split in half and used as input for two step-1 PCR reactions.