PHF3 regulates RNA stability through PHD and TLD domains - m6A
Three biological replicates of HEK293T WT, PHF3 KO and PHF3 ∆SPOC cells grown in 15 cm dishes were harvested at ~80% confluency, pellets were resuspended in 1.5 mL TRI reagent (Sigma) and incubated for 5 min at room temperature 300 µL chloroform (Applichem) was added and the lysate was vortexed and centrifuged at max. speed (21130xg) for 15 min at 4°C. The aqueous layer was transferred to a new tube and precipitated with 0.75 mL isopropanol. The RNA pellet was isolated by centrifugation for 30 min at 4°C, washed with 1 mL 75% ethanol, re-centrifuged for 10 min, dried and resuspended in 100 µL RNase free water. 120 µg RNA was treated with 240 U DNaseI (Roche) for 30 min at 37°C and subsequently purified by phenol-chloroform extraction and ethanol precipitation.  m6A-eCLIP, library preparation and sequencing was performed by Eclipse Bioinnovations (San Diego, USA).