Fig. 1

An overview of translatomic techniques. (i) Polysome profiling: mRNAs, 40S and 60S ribosomal subunits, monosomes, and polysomes are separated through sucrose gradient ultracentrifugation. Fractions containing polysomes are collected for RNA or protein analysis; (ii) TRAP-seq: through genetic modification, ribosomes of interest are labeled with affinity tags, which are under the control of a tissue-specific promoter. Subsequently, the labeled ribosomes with translating mRNAs can be caught by specific antibodies for further analysis, such as RNA-seq or microarray, after depleting rRNAs; (iii) Ribosome profiling: cell lysates are digested by RNase, which cleaves RNA regions that are not protected by ribosomes. Next, after depleting the contaminating rRNAs, RPFs are analyzed through deep sequencing to generate ribosome profiles, providing precise and abundant positional and quantitative information of ribosomes on translating mRNA; (iv) RNC-seq: Different from polysome profiling, translating mRNAs associated with ribosomes are separated through ultracentrifugation in a 30% sucrose cushion. This aids in RNC recovery, and the recovered RNC-mRNAs can preserve full-length information. (v) scRibo-seq: FACS-sorted single cells are lysed with a buffer supplemented with CHX and digested with micrococcal nuclease (MNase) to release RPFs, which are then converted into sequencing libraries by ligating adaptors that contain priming sites and a unique molecular identifier (UMI). Finally, inserts with typical RPF length were size-selected for deep sequencing. The strengths and drawbacks of each technique are summarized in the lower part of Fig. 1