GSM6965662: Ribo-seq_MSC, mutant#2, rep2; Homo sapiens; OTHER
Cell media was aspirated and cells were washed with ice-cold PBS. After removing PBS thoroughly, the dish was added 400 uL, which was then placed on wet ice for 5 min. Cells were then scraped off to a 1.5 mL tube and added NP40 (10%) for complete lysis for 10 min. The lysate was cleared by centrifugation at 2,000 g 4 °C for 10 min and then 20,000 g 4°C for 10 min. Lysate RNA was measured with Qubit HS RNA kit and certain amount of lysates were used for digestion at concentration (600 ng RNase A + 75 U RNase T1/ug RNA) at 25°C for 30 min. Stop the reaction by chilling on ice and add 50 U SUPERase In RNase inhibitior (2.5 uL/sample) and 0.5 M EDTA (100x). Following nuclease digestion, monosomes were purified using ultracentrifugation in a SW55i rotor at 55,000 rpm at 4°C for 5 h. PNK treatment was performed after pellet RNA recovery. Then 15% polyacrylamide TBE-Urea gel was used to isolate RPFs (26-34 nt) and the RPFs were recovered for library preparation. Library was prepared using optimized PARIS protocol (Lu. et al. Cell, 2016). The step of ribosomal rRNA depletion was performed after circularization step using pre-synthesized biotinylated probes whose sequences were from the protocol (Ingolia. et al. Nature protocol, 2012).