RAP-RNA

RAP-RNA

RNA sequencing of RAP isolates is different from standard RNA-Seq due to the noise from antisense RNA probes that were added. The complex is initially reverse-crosslinked and treated with DNase. The RNA mixture is dephosphorylated and ligated with an adaptor before reverse transcription. While the RNA-cDNA complexes are still annealed, the RNA probes are pulled-down with streptavidin to separate the cDNA fragments of interest from the antisense probes. After purification of cDNA fragments, a second adaptor is ligated and amplified through PCR before sequencing.

Pros:

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

Cons:

• Requires RNA sequence to be known beforehand

• RAP-RNA: 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. Boque-Sastre R., Soler M., Oliveira-Mateos C., Portela A., Moutinho C., et al. (2015) Head-to-head antisense transcription and R-loop formation promotes transcriptional activation. Proc Natl Acad Sci U S A 112: 5785-5790
2. McHugh C. A., Chen C. K., Chow A., Surka C. F., Tran C., et al. (2015) The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature
3. Engreitz J. M., Sirokman K., McDonel P., Shishkin A. A., Surka C., et al. (2014) RNA-RNA interactions enable specific targeting of noncoding RNAs to nascent Pre-mRNAs and chromatin sites. Cell 159: 188-199