A Single-copy Knock In Translating Ribosome ImmunoPrecipitation (SKI TRIP) tool kit for tissue specific profiling of actively translated mRNAs in C. elegans.. 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

翻译核糖体亲和纯化（Translating Ribosome Affinity Purification，TRAP）现已成为特异性分析特定细胞与组织类型中活跃翻译转录本的有力研究手段。将表位标记的核糖体亚基在特定细胞群中表达，可用于富集捕获核糖体及其结合的信使RNA（messenger RNA，mRNA），从而精准捕捉该时空条件下细胞类型特异性的翻译状态。当前秀丽隐杆线虫（Caenorhabditis elegans，C. elegans）研究领域可用的TRAP工具包，均基于随机整合至基因组的多拷贝阵列构建而成。

本研究介绍一种单拷贝敲入翻译核糖体免疫沉淀（Single-copy Knock In Translating Ribosome ImmunoPrecipitation，SKI TRIP）工具包：该工具包含一系列经CRISPR基因编辑构建的秀丽隐杆线虫品系，其基因组特定位点以单拷贝形式携带的表达盒，可驱动FLAG标记的核糖体亚基蛋白RPL-22在特定组织中表达。

对SKI TRIP品系及实验方法的深度表征显示，从内源基因座或特定细胞类型中表达的3×FLAG标记RPL-22，可整合进入活跃翻译的核糖体中；该工具能够高效且特异性地富集捕获细胞类型特异性转录本，且不会影响整体mRNA翻译过程或线虫的生存活力。我们建议将SKI TRIP工具用于研究对翻译变化极为敏感的生物学过程，例如衰老。

实验整体设计：对野生型（Wild Type，WT，所有多聚核糖体的多聚核糖体谱分析RNA）及MSD470泛表达型品系的秀丽隐杆线虫进行多聚核糖体谱分析（Polysome Profiling）后开展RNA测序（RNA sequencing，RNAseq），每个品系设置三次生物学重复，分别获取总RNA与多聚核糖体谱分析RNA；其中MSD470品系额外获取1份所有多聚核糖体的多聚核糖体谱分析RNA（PP）、1份60S、80S及多聚核糖体组分的多聚核糖体谱分析RNA（PP2）。同时，对MSD470（泛表达型，UBI）、WBM1364（体细胞型）、WBM1471（神经B型）、MSD473（神经A型）、WBM1339（肌肉型）及WBM1471（肠道型）等品系的秀丽隐杆线虫开展SKI TRIP免疫沉淀（Immunoprecipitation，IP）实验后进行RNA测序，每个品系同样设置三次生物学重复，分别获取总RNA与免疫沉淀富集的RNA。