GSM7400803: TTseq.12h.rep3-1; Mus musculus; OTHER
TT-seq was performed as previously described (Schwalb et al. Science 2016. 352, 1225-1228) with slight modifications. Cells were labeled with 4SU (Glentham Life Sciences, GN6085) at 1mM final concentration for 10 minutes in 37 °C incubator. The medium was then immediately decanted, and cells were lysed by directly adding 5 ml Trizol reagent onto the plates. Cells were scraped from the plates and collected in 15 mL falcon tubes and frozen in -80 °C. Total RNA were extracted using Trizol RNA extraction protocol following the manufacturer's instructions and stored in -80 °C. S. cerevisiae 4-thiouracil (4TU, Sigma, 440736) labelled RNAs were prepared following the protocol described previously (Gregersen et al. Nature Protocols 2020. 15, 604-627). 110 μg total RNA, combined with 1 μg of spike-in S. cerevisiae 4TU-labeled RNA were fragmented with 166 mM NaOH for 30 minutes on ice, and stopped by adding 400 mM Tris-HCl, pH 6.8 and passed through Micro Bio-Spin P-30 gel columns twice. The total RNAs were then denatured at 65°C for 10min, followed by 5mins of incubation on ice, and mixed with 50 μL of 0.1 mg/ml MTSEA biotin-XX linker (Biotium, BT90066) on rotator at room temperature for 30 minutes in the dark. The RNA was then purified with phenol/chloroform/isoamyl alcohol (25:24:1 (vol/vol/vol)), followed by isopropanol precipitation. The RNA was then resuspended in nuclease-free H2O and denatured at 65°C for 10min, followed by 5 minutes of incubation on ice and mixed with 150 μL of μMACS streptavidin MicroBeads (Miltenyi, 130-074-101) for 15 minutes in cold room on a rotating wheel. The biotinylated RNA was retained on μMACS magnetic separator (Miltenyi, 130-042-602) placed on a MACS multistand (Miltenyi, 130-042-303) and washed three times with 900 μL of pre-warmed (65 °C) pull-down wash buffer (100 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1 M NaCl and 0.1% Tween 20) and three more times with 900 μL of room temperature pull-down wash buffer. The biotinylated RNA was eluted twice from the column with 100 μl of 100 mM DTT and purified with RNeasy MinElute Cleanup Kit (Qiagen, 74204).