GSM4877164: PRO-Seq_RUVBL2_Auxin_1h_rep2; Mus musculus; OTHER
Total RNA were extracted using Trizol reagent, and nascent RNA were enriched with biotin according to the protocol described by Mahat et al., 2016. The enriched RNAs were reverse transcribed on beads, simply, SuperScript IV RT buffer included N9 random primers were added into the RNA bound beads to synthesis first strand cDNA on beads according to user's manual, the excess RT primers were removed by addition of 3 μl Exonuclease I (Exo I) (NEB, M0293S), digested for 1h at 37 °C. The RNA was eliminated by adding 2 µl of 1M NaOH and incubating for 20 min at 98 °C. The reaction was then neutralized with 2 µl 1M HCl, the released cDNAs were extracted using Tris saturated equilibrium phenol (pH 8.0). Finally, the purified cDNA was subjected to library preparation according to the TELP protocol (Peng et al., 2015), the 150~400 bps length amplified DNA were selected for library analysis and finally subject to HiSeq X-ten PE150 sequencing (Novogene, Beijing). Each condition performed two technical duplicates.