GSM8565425: ZCCHC8_FKBP12^F36V, Z12H clone, Ctrl, 0min, rep2; Mus musculus; RNA-Seq
When reaching 60 to 70% confluency, cells were either untreated or dTAG-13 treated for 2 hours, followed by additional 1 μg/ml doxycycline induction for 20 hours. Then, transcribed RNA was labelled with 4sU by incubation with medium containing 500 μM 4sU (Lexogen) and 1 μg/ml doxycycline for another 4 hours. Next, 4sU was withdrawn for all samples by medium washout. Different samples were washed with medium containing 1 μg/ml doxycycline and 50 mM uridine (in excess, Lexogen) for 1.5 and 3 hours. Cells are quickly washed with PBS once and directly lysed with Trizol/chloroform for total RNA isolation. Equal amount of RNA samples (5 μg) was treated with Iodoacetamide to modify the 4-thiol group of S4U-containing nucleotides via the addition of a carboxyamidomethyl group by the SLAMseq Kinetics Kit. The RNA was recovered using RNAClean XP beads (Beckman Coulter), followed by resuspension in nuclease free water. Approximately 500 ng RNA of each sample was taken forward for library preparation using the Illumina TruSeq stranded total RNA kit. Quantification of the libraries was conducted by qPCR using KAPA Library Quantification DNA standards. Finally, the libraries were pooled and paired-end sequencing was performed using Illumina NextSeq500.