GSM7943595: 30min Torin1 treatment biological replicate 3; Mus musculus; OTHER
RNA samples were harvested using TRIzol reagent (invitrogen) from cells metabolically labeled with 500 mM 4sU exactly at 10min to the end of the given length of treatments. RNA samples then were treated to remove residual DNA by using RNeasy Micro Kit (Qiagen, 74004) combined with Amplification Grade DNase I (18068015, Invitrogen) and Superase-In RNase inhibitor (AM2694, Thermo Fisher Scientific). RNA samples next were chemically fragmented by cold 5 × fragmentation solution at 94°C for 3.0 minutes and biotinylated as described in Duffy et al. (2015). After removing of the excess biotin, biotinylated RNA was heated at 65°C for 5min, placed on ice for 2 min, and mixed with 50 µl of Dynabeads MyOne Streptavidin C1 (Invitrogen, 65001). The Dynabeads engaged biotinylated RNA was eventually eluted and purified using Norgen RNA clean-up and concentration MicroElute kit (Norgen #61000) and for library construction. Nascent RNA libraries for TT-seq were prepared using TruSeq RNA Library Preparation Kit v2, Set A and B following manufacturer's protocol.