GSE195601
A Single-copy Knock In Translating Ribosome ImmunoPrecipitation (SKI TRIP) tool kit for tissue specific profiling of actively translated mRNAs in C. elegans.
Translating Ribosome Affinity Purification (TRAP) methods have emerged as a powerful approach to profile actively translated transcripts in specific cell and tissue types.  Epitope tagged ribosomal subunits are expressed in defined cell populations and used to pull down ribosomes and their associated mRNAs, providing a snapshot of cell type-specific translation occurring in that space and time. Current TRAP toolkits available to the C. elegans community have been built using multi-copy arrays, randomly integrated in the genome.  Here we introduce a Single-copy Knock In Translating Ribosome ImmunoPrecipitation (SKI TRIP) tool kit, a collection of C. elegans strains engineered by CRISPR in which tissue specific expression of FLAG tagged ribosomal subunit protein RPL-22 is driven by cassettes present in single copy from defined sites in the genome. In depth characterization of the SKI TRIP strains and methodology shows that 3xFLAG tagged RPL-22 expressed from its endogenous locus or within defined cell types incorporates into actively translating ribosomes and can be used to efficiently and cleanly pull-down cell type specific transcripts without impacting overall mRNA translation or fitness of the animal. We propose SKI TRIP use for the study of processes that are acutely sensitive to changes in translation, such as aging. Overall design: RNAseq from C. elegans after Polysome Profiling (total RNA and polysome profiling RNA in triplicates per strain) from WT (polysome profiling RNA of all polysomes) and strains MSD470=ubiquitous (1x polysome profiling RNA of all polysomes (PP); 1x polysome profiling RNA of 60S, 80S and polysomal ribosomes (PP2)) RNAseq from C. elegans after SKI TRIP (IP) experiments (total RNA and IPed RNA in triplicates per strain) from strains MSD470=UBI, WBM1364=soma, WBM1471=neuroB, MSD473=neuroA, WBM1339=muscle, WBM1471=intestine