Dunn-Davis et al. Combined snoRNA interaction lists
Description
Dataset 1 from Dunn-Davis et al.: Systematic mapping of small nucleolar RNA targets in human cells. (Doi: 10.1101/2021.07.22.451324). Spreadsheet listing all recovered RNA-RNA interaction for human snoRNAs.
Files
Steps to reproduce
Systematic mapping of box C/D snoRNA interactions by UV crosslinking To identify potential novel snoRNA interactions we initially applied the CLASH technique. which involves UV crosslinking of RNA complexes with tagged proteins in living cells and tandem-affinity purification of the RNP complexes under stringent conditions. Ligation of linker adaptors is performed in parallel with internal ligation of captured RNA fragments base paired to each other. RNA is isolated, followed by reverse transcription and high throughput sequencing of cDNA libraries. Sequence data is then mapped to the genome. Most reads map to a single genomic site (termed ‘single reads’), representing the position at which the tagged protein was crosslinking to the transcriptome. However, if the crosslinked RNA forms part of a stable duplex, the ends of the two RNA species can be ligated together forming a chimeric cDNA. These are identified when two different regions of the resulting cDNA sequence map robustly to distinct sites in the genome (termed ‘hybrid reads’). CLASH analyses require the use of a “bait” protein fused to a tandem affinity purification tag. For these analyses, we tagged the core box C/D snoRNP proteins FBL or NOP56 with a C-terminal tag consisting of His6 – Precision protease cleavage site – Flag epitope. Orthogonal analyses used formaldehyde assisted crosslinking ligation and sequencing of hybrids (FLASH). This approach is similar to CLASH in that RNA-protein interactions are captured by UV crosslinking in growing cells, but antibodies are used for immunopurification of endogenous RNA-protein complexes instead of tagged constructs. During purification, brief formaldehyde crosslinking is used to stabilize binding of the covalent bait protein-RNA complex to the protein A beads, allowing column washes under highly denaturing conditions In human cells we recovered 591,958 hybrids overall (Dataset 1). Recovered sequences that could be confidently mapped to two distinct regions of the genome (see Methods) were regarded as representing chimeric cDNAs resulting from RNA-RNA ligation. Non-identical chimeric sequences, or sequences recovered from different analyses, in which both segments overlapped were regarded as demonstrating independent recovery of the same interaction.
Institutions
- The University of Edinburgh