title: PHF3 regulates RNA stability through PHD and TLD domains - RNAseq PHD - TLD
design: PHF3 regulates RNA stability through PHD and TLD domains - RNAseq PHD - TLD
library_construction_protocol: Cells from a 90% confluent 10 cm dish were harvested and counted, 8 million cells were mixed with 2 million Drosophila S2 cells as a spike-in control. Cell pellets from one well of a 6-well plate were resuspended in 1 mL TRI reagent (Sigma) and incubated for 5 min at room temperature. 200 µL chloroform (Applichem) was added and the lysate was vortexed and centrifuged at max. speed (21130 g) for 15 min at 4°C. The aqueous layer was transferred to a new tube and precipitated with 0.5 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 70 µL RNase free water. 20 µg RNA was treated with 40 U DNaseI (Roche) for 30 min at 37°C and subsequently purified by phenol-chloroform extraction and ethanol precipitation. rRNA was depleted using RiboCop rRNA depletion kit (Human/Mouse/Rat) (Lexogen) and libraries were prepared using Corall total RNA Seq kit (Lexogen) according to the manufacturer's instructions, starting with 600 ng total RNA input.