SPRITE uses a split–and–pool strategy to uniquely barcode all molecules within a crosslinked complex by repeatedly splitting all complexes into a 96–well plate, ligating a specific tag sequence within each well, followed by pooling of these complexes such that the final product contains a series of tags ligated to each molecule, which we refer to as a barcode.
Watch the Guttman Lab SPRITE video protocol for step by step instructions:
CLAP integrates an epitope tag into a protein of interest which enables covalent coupling of the protein to a resin (e.g. HaloTag, SpyTag) thereby enabling a purification with fully denaturing conditions – including high temperatures, high concentrations of denaturants and detergents, and chaotropic salts – that disrupt protein and RNA folding.
To achieve high specificity, RAP utilizes 120-nucleotide antisense RNA probes in order to form extremely strong hybrids with the target RNA thereby enabling purification using denaturing conditions that disrupt nonspecific RNA-protein interactions and nonspecific hybridization with RNAs or genomic DNA. To achieve high resolution, RAP uses DNase I to digest genomic DNA to ~150bp fragments, which provides high resolution mapping of binding sites. To robustly capture a lncRNA, RAP uses a pool of overlapping probes tiled across the entire length of the target RNA to ensure capture even in the case of extensive protein-RNA interactions, RNA secondary structure, or partial RNA degradation.
The RAP-MS method uses long biotinylated antisense probes, which form very stable RNA-DNA hybrids, and therefore can be used to purify lncRNA complexes in denaturing and reducing conditions. The RAP-MS method has been optimized to achieve higher yields and enrichment levels of the endogenous RNA complexes relative to our original protocol used for DNA and RNA. To achieve sensitive quantification and to distinguish between specific proteins and background proteins, RAP-MS uses Stable Isotope Labeling by Amino acids in Culture (SILAC) to label proteins and specific lncRNA captures are directly compared to a control ncRNA.
RNAtag-Seq is a method for generating a single RNA-Seq library from a large number of independently barcoded RNA samples. This method provides highly reproducible, strand-specific, quantitative sequencing covering the full lengths of transcripts in diverse species. This approach dramatically increases the throughput and decreases the cost per sample of RNA-Seq library construction.