GSM7124088: Run 1 WT input replicate 1; Homo sapiens; OTHER
Cells were flash frozen by submerging the bottoms of dishes in liquid nitrogen. Cells were collected and lysed by scraping in lysis buffer containing cycloheximide. Cell lysates were triturated and clarified by centrifugation. Input samples were extracted from aliquot of lysate using trizol reagent according to the manufacturer's protocol. Following phase separation, the aqueous phase was applied to a Qiagen RNAeasy column and purified according to kit instructions. For ribosome prototected fragments, lyates were treated with RNase I and loaded onto 15%–45% sucrose gradients. Samples were fractionated and fractions containing 80S monosomes were precipitated with ethanol. After precipitation, monosome containing fractions were resuspended in trizol reagent. Sample workup was performed with a Zymo direct-zol kit following the manufacturer's protocol. Samples were ribodepleted using Ribopoo ribosome RNA Homo sapiens depletion kit supplemented with custom-made oligos. RPF and input fractions were fragmented and size-selected on denaturing Urea PAGE gels. 3' adaptors sequencing adaptors were added with RNA ligase and reactions were reverse-transcribed with SuperScript IV. Sample cDNA was circularized with circLigase and libraries were PCR amplified to add Illumina sequencing primers. Libraries were sequenced on a NextSeq 2000 P2 instrument.