GSM7184768: MEL624, cytokine-treated, L22-HA_PD, rep2; Homo sapiens; OTHER
Cells were treated with 200 μg/ml cycloheximide for 3-5 minutes at 37 °C and cell pellets were washed with ice-cold PBS containing 200 μg/ml cycloheximide. Subsequently, pellets were lysed for 30 minutes in ice-cold lysis buffer (20 mM Tris HCl pH 7.4, 10 mM MgCl2, 150 mM KCl, 1% NP-40, 200 µg/mL cycloheximide and 1x EDTA-free proteinase inhibitor cocktail (Sigma 5892791001). Lysates were centrifuged at 12.000 x g for 20 minutes at 4 °C and the supernatants were collected for RNA digestion using RNaseI (Thermo Scientific AM2294) for 40 minutes at 25 °C. RNA digestion was stopped using SUPERase (Thermo Scientific AM2694). Digested lysates were incubated with Pierce™ Control Agarose Matrix (Thermo Scientific 26150) for 15 minutes at 4°C followed by incubation with AntiHA.11 Epitope Tag Affinity Matrix (BioLegend 900801) for 4 hours at 4°C under constant rotation. Supsequely, tagged ribosomes were eluted with 1mg/ml HA peptide (Thermo Scientific 26184) for 15 minutes at 30°C. Finally, ribosome protected fragments (RPFs) were purified using miRNeasy minikit (Qiagen) according to manufacturer's instructions and RNA concentrations were measured with the Agilent 2100 bioanalyzer using a Small RNA Chip (Agilent 5067-1548). Briefly, RPFs were run on a 15% TBE-urea polyacrylamide gel and size-selected between 26nt and 32nt using RNA markers. RNA was extracted using RNA gel extraction buffer (300 mM NaOAc pH 5.5, 1.0 mM EDTA, 0.25% SDS) and precipitation for 30 minutes on dry ice. After precipitation, samples were incubated overnight at 25°C. End dephosphorylation was performed using T4 polynucleotide kinase (PNK) (NEB M0201) for 1 hour at 37°C and ligation was performed using the T4 RNA ligase I (NEB M0204) for 2 hours at 25°C. Reverse transcription was performed using the SuperScript III reverse transcriptase (Thermo Scientific 18080051) using the oCJ485 RT primer for 30 minutes at 50°C. cDNA was then size-selected at 110bp using a 10% TBE-urea polyacrylamide gel. DNA was extracted using DNA gel extraction buffer (300 mM NaCl, 10 mM Tris pH 8, 1 mM EDTA) and precipitation for 30 minutes on dry ice. After precipitation, samples were incubated overnight at 25°C. Circular ligation was performed using CircLigase II ssDNA Ligase (Epicentre CL4111K) for 1,5 hours at 60°C. cDNA amplification was performed for 15 cycles with the Phusion High-Fidelty DNA polymerase (Thermo Scienfific F530) and primers containing different indexes for sequencing. The amplification product was size-selected at 180bp using a 8% TBE polyacrylamide gel. Samples were measured with the Agilent 2100 bioanalyzer using a DNA chip (Agilent 5067-1506) to assure high quality followed by molarity quantification. Sequencing was performed on the Illumina HiSeq2500.