CapSeq
CapSeq
CapSeq maps potential transcription start sites . In this method, total RNA is treated with terminator 5′-phosphate-dependent exonuclease to degrade rRNAs, calf intestinal phosphatase (CIP) to remove 5′ phosphates, and tobacco acid pyrophosphatase (TAP) to remove 5′ caps. The resulting long-capped RNAs are ligated to a 5′ adaptor. The first-strand cDNA is primed using a pool of random octamers and sequenced after size selection. Sequencing helps determine potential transcription start sites for PolII loci.
Pros:
- Maps RNAs anchored to RNA polymerase II
Cons:
- Multiple steps and treatments can lead to loss of material
- Tobacco Acid Pyrophosphatase (TAP)
- Calf Intestinal Phosphatase (CIP)
- APex Heat-Labile Alkaline Phosphatase
- Hainer S. J., Gu W., Carone B. R., Landry B. D., Rando O. J., et al. (2015) Suppression of pervasive noncoding transcription in embryonic stem cells by esBAF. Genes Dev 29: 362-378
- Tsai H. Y., Chen C. C., Conte D., Jr., Moresco J. J., Chaves D. A., et al. (2015) A ribonuclease coordinates siRNA amplification and mRNA cleavage during RNAi. Cell 160: 407-419
- Mohn F., Sienski G., Handler D. and Brennecke J. (2014) The rhino-deadlock-cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila. Cell 157: 1364-1379
- Hainer S. J., Gu W., Carone B. R., Landry B. D., Rando O. J., et al. (2015) Suppression of pervasive noncoding transcription in embryonic stem cells by esBAF. Genes Dev 29: 362-378
- Tsai H. Y., Chen C. C., Conte D., Jr., Moresco J. J., Chaves D. A., et al. (2015) A ribonuclease coordinates siRNA amplification and mRNA cleavage during RNAi. Cell 160: 407-419
- Mohn F., Sienski G., Handler D. and Brennecke J. (2014) The rhino-deadlock-cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila. Cell 157: 1364-1379