GSM4731596: 5890_4_sgNT1_IFNy_ACAGTG_S73_R1_001; Homo sapiens; OTHER
1x106 MD55A3 cells were seeded. Cells were rinsed once with PBS and the appropriate medium was added for exactly 48h. After 48h cells were harvested and ribosome profiling was performed based on  Ingolia NT et al, doi:10.1038/nprot.2012.086 (2012). Adjustment to this protocol are as follows: 20μL of fresh cycloheximide (100g/mL) was added to the medium and plates were incubated at 37°C for 1min. Cells were washed twice with PBS containing CHX and harvested into Lysis buffer as described. Samples were centrifuged at 4°C full speed for 10min and the cell pellet was snap frozen in liquid nitrogen. 400µL of lysate was incubated with 3µL of RNAse I (Ambion AM2295 100 U/µL) for 45min at RT in an Eppendorf shaker, 600RPM. Then, the digestion was inactivated by adding 12µL of SUPERaseInhibitor (AM2696, ThermoFisher). Ribosomes were pelleted as described by ultra-centifugation using TLA 100.3 rotor for 1h30, 4°C at 100,000rpm. The pellet was retrieved in 1mL Trizol (Life Technologies). RNA was extracted using manufacturer's instruction. RNA pellet was resuspended with 15L of 10mM Tris pH-7.  15% TBE-urea gel was pre-run for 20min at 200V. 10µg of RNA maximum was loaded per lane. 2X gel loading buffer was used (AM8547, Invitrogen). 10µM oligos marker of 26nt and 32nt were used as size selection markers. RNA was extracted as described in the protocol using the Rapid Gel extraction. RNA was then precipitated overnight using Isopropanol (Sigma) and Glycoblue (AM9516, ThermoFisher). The construction of ribosome protected fragments (RPF) libraries were done as previously described in Ingolia NT et al, doi:10.1038/nprot.2012.086 (2012). Possible changes are as follows: For linker ligation, RNA was resuspended into 8µL of 10mM Tris pH 7.0. Then, 1µL of PNK buffer and 1µL of T4 PNK (M0201L, NEB) were added. Samples were incubated for 1h at 37°C. Then, 4µL of 50% PEG-800, 0.5µL of 10X Ligase buffer, 0.2µL of Universal miRNA cloning linker (S1315S, NEB) and 0.75µL of RNL2 Ligase (M0351S, NEB) were added. Samples were incubated for 3h at 25°C. Finally, 0.5µL of Deadenylase (M0331S, NEB) and 2µL of RecJ exonuclease (M0264L, NEB) were added. Samples were incubated at 30°C for 1. RNA was then precipitated overnight as previously described. RNA was resuspended in 6,5µl 10mM Tris pH 7.0 and mixed with 0.75µL of 10X dNTPs and 0.5µL of Reverse transcription primer (50µM solution). Samples were incubated 5min at 65°C. Then, Reverse transcription was performed using SuperScript III RT (18080-051, Life technologies) using manufacturer's instruction. Then, as described the reaction was separated using and 10% TBE-urea gel and DNA was extracted from gel and precipitated overnight. Then, as described the reaction was separated using and 10% TBE-urea gel and DNA was extracted from gel and precipitated overnight.  Circularization was as described using CircLigase II ssDNA Ligase (CL9021K, epicentre)