GSM4669098: Ythdc1_cKO_ESC#2_+4OHT.8min.rep2; Mus musculus; OTHER
total RNA was extracted using miRNeasy mini kit (Qiagen #217004) with RNase-free DNase set (Qiagen, #79254) Metabolic labeling nascent RNA with 4sU and nascent RNA purification was performed as described4. In brief, Ythdc1 cKO ESCs were pre-treated with DMSO or 4OHT (1 μg/ml) for 4 days, then incubated with 100 μM DRB (Sigma, #D1916) for 3 h to block Pol II transcription. Cells were washed 3 times with PBS to remove DRB and recover transcription, and newly transcribed RNA was labeled with 1 mM 4sU (Sigma, #T4509) for 0, 4, 8 min. Transcription was stopped by adding TRizol (Invitrogen) into the cells and total RNA was extracted using miRNeasy mini kit (Qiagen, #217004) with RNase-free DNase set (Qiagen, #79254). 100 μg total RNA was used for biotinylation and purification of 4sU-labeled nascent RNA. 4sU labeled RNA was incubated with Biotin HPDP-DMF stock (Dissolve 50 mg of EZ-Link HPDP-biotin (Thermo Scientific, #21341) in 50 ml of DMF (Sigma, # D4551)) in biotinylation buffer (10 mM Tris pH 7.4, 1 mM EDTA) for 2 h on a tube rotator at room temperature. Unbound HPDP-biotin was removed by equal volume chloroform extraction twice, and RNA was precipitated at 14,000 rpm for 30 min at 4°C with 1:10 volume of 5 M NaCl and an equal volume of isopropanol. The RNA pellet was washed twice with 75% ethanol and resuspended in 100 μl of nuclease-free water. Biotinylated RNA was enriched by 100 μl Streptavidin T1 (Invitrogen, #65601) at room temperature for 20 min. Beads were washed five times with 1 ml washing buffer (100 mM Tris pH 7.4, 10 mM EDTA, 1 M NaCl, 0.1% Tween 20). Nascent RNA was eluted with 100 μl 100 mM dithiotheitol (DTT, Thermo Scientific, #A39255) twice and was purified with the RNeasy MinElute clean-up kit (Qiagen, #74204) according to the manufacturer's instructions.
GEO Accession
GSM4669098