With the advent of the high-throughput DNA and RNA sequence identification methods (such as Illumina sequencing), it became possible to efficiently analyse nucleotide sequences of large numbers of relatively short DNA and RNA fragments. Sequences of these fragments can be superimposed to reconstruct the source. Alternatively, if the source sequence is already known, the fragments can be found within it (“mapped”), and their individual numbers counted. Thus, if an initial stage exists whereby the fragments are differentially present or selected (“enriched”), this approach can be used to quantitatively describe such stage over even a very large number or length of the input sequences, most usually encompassing the entire DNA or RNA of the cell.
TCP-seq is based on these capabilities of the high-throughput RNA sequencing and further uses the nucleic acid protection phenomenon. The protection is manifested as resistance to depolymerisation or modification of stretches of nucleic acids (particularly, RNA) that are tightly bound to or engulfed with other biomolecules, which thus leave their “footprints” over the nucleic acid strand. These “footprint” fragments therefore represent location on nucleic acid chain where the interaction occurs. By sequencing and mapping the fragments back to the source sequence, it is possible to precisely identify the locations and counts of these intermolecular contacts.
In case of TCP-seq, ribosomes and ribosomal subunits engaged in interaction with mRNA are first fast chemically crosslinked to it with formaldehyde to preserve existing state of interactions (“snapshot” of distribution) and to block any possible non-equilibrium processes. The crosslinking can be performed directly in, but not restricted to, live cells. The RNA is then partially degraded (e.g. with ribonuclease) so that only fragments protected by the ribosomes or ribosomal subunits are left. The protected fragments are then purified according to the sedimentation dynamics of the attached ribosomes or ribosomal subunits, de-blocked, sequenced and mapped to the source transcriptome, giving the original locations of the translation complexes over mRNA.
TCP-seq merges several elements typical to other transcriptome-wide analyses of its kind. In particular, polysome profiling and ribosome (translation) profiling approaches are also employed to identify mRNA involved in polysome formation and locations of elongating ribosomes over coding regions of transcripts, correspondingly. These methods, however, do not use chemical stabilisation of translation complexes and purification of the covalently bound intermediates from the live cells. TCP-seq thus can be considered more as a functional equivalent of ChIP-seq and similar methods of investigating momentary interactions of DNA that are redesigned to be applicable for translation.