RAP

RAP

RAP isolates lncRNAs and maps the sequence of their target DNA through a probe-capture mechanism. First, the cells are crosslinked and lysed before DNase I chromatin digestion to 100–300 bp DNA fragments. Biotinylated RNA probes, antisense to the lncRNA, are hybridized and captured with streptavidin. The biotin-RNA probes are 120 nt and are tiled every 15 nt over the span of the lncRNA. The captured complexes are eluted and prepared for sequencing. RNA library preparation is done through RAP-RNA, and DNA library preparation by standard chromatin immuniprecipitation (ChIP).

Pros:

• Genomic mapping of lncRNA targets
• Possible to sequence RNA and DNA from the purification products
• Long RNA probe length provides high binding affinity to the target lncRNA¹
• Minimal amplification steps during RNA sequencing after purification of the lncRNA complex

Cons:

• Requires RNA sequence to be known

• RAP: Engreitz J. M., Pandya-Jones A., McDonel P., Shishkin A., Sirokman K., et al. (2013) The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome. Science 341: 1237973
• 1. Kashi K., Henderson L., Bonetti A. and Carninci P. (2015) Discovery and functional analysis of lncRNAs: Methodologies to investigate an uncharacterized transcriptome. Biochim Biophys Acta

1. Kashi K., Henderson L., Bonetti A. and Carninci P. Discovery and functional analysis of lncRNAs: Methodologies to investigate an uncharacterized transcriptome. Biochim Biophys Acta. 2015;
2. Simon M. D. Insight into lncRNA biology using hybridization capture analyses. Biochim Biophys Acta. 2016;1859:121-127

1. Chen C. K., Blanco M., Jackson C., et al. Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing. Science. 2016;
2. Boque-Sastre R., Soler M., Oliveira-Mateos C., et al. Head-to-head antisense transcription and R-loop formation promotes transcriptional activation. Proc Natl Acad Sci U S A. 2015;
3. McHugh C. A., Chen C.-K., Chow A., et al. The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature. 2015;521:232-236
4. Engreitz J. M., Sirokman K., McDonel P., et al. RNA-RNA interactions enable specific targeting of noncoding RNAs to nascent Pre-mRNAs and chromatin sites. Cell. 2014;159:188-199