CHART
Capture Hybridization Analysis of RNA Targets
CHART maps genomic binding sites of ncRNAs by isolating and sequencing the DNA regions where the crosslinked RNA-DNA-protein complexes are bound (Simon et al., 2011). CHART differs from other crosslinked-complex purification techniques, such as ChIRP, due to the use of biotinylated 24 nt oligonucleotides (C-oligos) that are highly sensitive and unique to the ncRNA of interest (Kashi et al., 2015). An RNase H mapping assay is used to design the 24 nt sequence of the C-oligos. First, nuclei samples are crosslinked and fragmented. Next, C-oligos are hybridized to the complex and bound to streptavidin beads. The mixture is washed and the complex eluted. The DNA is isolated and sequenced, and the proteins involved in the complex are isolated and analyzed by Western blots.
Advantages:
- Maps genomic binding sites of lncRNAs
- Simultaneously identifies proteins associated with the lncRNA complex
Disadvantages:
- Needs large amount of nuclei (1 x 10^9 cells)
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Hassan M. Q., Tye C. E., Stein G. S. and Lian J. B. Non-coding RNAs: Epigenetic regulators of bone development and homeostasis. Bone. 2015;81:746-756
References:
Lee N., Moss W. N., Yario T. A. and Steitz J. A. EBV noncoding RNA binds nascent RNA to drive host PAX5 to viral DNA. Cell. 2015;160:607-618
Torres M., Becquet D., Blanchard M. P., et al. Circadian RNA expression elicited by 3′-UTR IRAlu-paraspeckle associated elements. Elife. 2016;5:
Vance K. W., Sansom S. N., Lee S., et al. The long non-coding RNA Paupar regulates the expression of both local and distal genes. EMBO J. 2014;33:296-311
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History: CHART
Revision by sbrumpton on 2017-06-21 07:50:23 - Show/Hide
Capture Hybridization Analysis of RNA Targets
CHART maps genomic binding sites of ncRNAs by isolating and sequencing the DNA regions where the crosslinked RNA-DNA-protein complexes are bound (Simon et al., 2011). CHART differs from other crosslinked-complex purification techniques, such as ChIRP, due to the use of biotinylated 24 nt oligonucleotides (C-oligos) that are highly sensitive and unique to the ncRNA of interest (Kashi et al., 2015). An RNase H mapping assay is used to design the 24 nt sequence of the C-oligos. First, nuclei samples are crosslinked and fragmented. Next, C-oligos are hybridized to the complex and bound to streptavidin beads. The mixture is washed and the complex eluted. The DNA is isolated and sequenced, and the proteins involved in the complex are isolated and analyzed by Western blots.
Advantages:- Maps genomic binding sites of lncRNAs
- Simultaneously identifies proteins associated with the lncRNA complex
Disadvantages:- Needs large amount of nuclei (1 x 10^9 cells)
Reagents:Illumina Library prep and Array Kit SelectorReviews:Hassan M. Q., Tye C. E., Stein G. S. and Lian J. B. Non-coding RNAs: Epigenetic regulators of bone development and homeostasis. Bone. 2015;81:746-756References:Lee N., Moss W. N., Yario T. A. and Steitz J. A. EBV noncoding RNA binds nascent RNA to drive host PAX5 to viral DNA. Cell. 2015;160:607-618Torres M., Becquet D., Blanchard M. P., et al. Circadian RNA expression elicited by 3'-UTR IRAlu-paraspeckle associated elements. Elife. 2016;5:Vance K. W., Sansom S. N., Lee S., et al. The long non-coding RNA Paupar regulates the expression of both local and distal genes. EMBO J. 2014;33:296-311