Optimized protocol for tRNA identification in the ribosomal complexes from human cell lines

Here, we describe a protocol for tRNA identification in the 60S ribosome-nascent peptide complex co-purified with Nuclear Export Mediator Factor (NEMF), a responsible factor for C-terminal alanine and threonine tailing of the nascent peptide. Our protocol is based on regular reverse transcription followed by quantitative Polymerase chain reaction (PCR). Although this method cannot distinguish between amino acid-charged and uncharged and base-modified and unmodified tRNAs, it is a convenient way to estimate the relative level of tRNA species and thus can be useful for researchers. For complete details on the use and execution of this protocol, please refer to Udagawa et al. (2021). [Display omitted] •Strategy to obtain the ribosomal complex from mammalian cells•Purification of the ribosomal complex with sucrose density gradient centrifugation•A simple protocol to quantify the levels of individual tRNA species by RT-qPCR Here, we describe a protocol for tRNA identification in the 60S ribosome-nascent peptide complex co-purified with NEMF, a responsible factor for C-terminal alanine and threonine tailing of the nascent peptide. Our protocol is based on regular reverse transcription followed by quantitative PCR. Although this method cannot distinguish between amino acid-charged and uncharged and base-modified and unmodified tRNAs, it is a convenient way to estimate the relative level of tRNA species and thus can be useful for researchers.