GSM8987277: Input RNA, WT control, replicate 1; Homo sapiens; OTHER
Ribosome profiling. Cells were washed briefly with ice-cold PBS (supplemented with 100 µg/ml Cycloheximide (CHX)) and the cell pellet was resuspended in RP-Lysis Buffer (20 mM Tris-HCL pH 7.5, 10 mM MgCl2, 100 mM KCl, 1 % Triton-X 100, 2 mM DTT, 100 µg/ml CHX, 1x EDTA-free Complete Protease Inhibitor Cocktail). After lysis, all samples were centrifuged at 6400 rpm, 5 min, 4 °C. The supernatant was digested with 1 U/µl of RNase I for 45 min at room temperature. Digested lysates were run through a 7 % - 47 % sucrose gradient using a Beckmann Coulter SW41 Ti rotor at 36,000 rpm at 4 °C for 2 h. Monosome fractions were obtained and digested with 1 % SDS and 0,15 µg/µl Proteinase K for 45 min at 45 °C. Resulting footprints were extracted following a standard Trizol-Chloroform extraction protocol and size-selected using a 12 % denaturing gel. Library construction: Footprint RNA was dephosphorylated using 5 U of T4 PNK for 30 min at 37°C. Subsequently, preadenylated UMI-linkers were ligated to the RNA 3'end using 100 U T4 RNA Ligase. Residual linker was eliminated with 25 U 5' Deadenylase and 15 U RecJf for 60 min at 30 °C. Ribosomal RNA was depleted using a biotinylated rRNA oligo pool in 1x SSC buffer (3 M NaCl, 300 mM trisodium citrate, pH 7) and pulled down using MyOne Streptavidin C1 DynaBeads. Resulting RNA footprints were reverse transcribed using the SuperScript III First-Strand Synthesis System. Resulting cDNA was size-selected using an 8 % denaturing gel. cDNA was circularized using the CircLigase II Kit from Lucigen. Samples were subjected to PCR to introduce Illumina i7 indexes, followed by size-selection on an 8 % non-denaturing PAGE gel. Resulting sample concentrations were measured with Qubit 3.0 using Qubit DNA HS kit. The final libraries were single-end sequenced with an Illumina NextSeq2000 P2 system.