GSM5412817: Ribo_PFB_Lib4_EGFP_rep2; Homo sapiens; OTHER
PFB cells and ground  mouse livers powder were lysed on ice in the presence of cycloheximide.  Lysates were clarified by centrifugation. For ribosome profiling, monosomes were generated by RNaseI digest followed by ultracentrifugation and purified by miRNeasy mini kit. Ribosome proteced RNA fragments ranging from 26-34 nt were gel purified. Upon dephosphorylation of sample RNA, linker was ligated and this product  was followed by reverse transcription and gel purification. Then the RT product was circularized by CircLigase ssDNA ligase followed by rRNA depletion with biotinylated subtraction oligo pool. Libraries were then PCR-amplified, and gel-purified libraries were pooled in equimolar ratios and submitted for sequencing. Ribosome profiling