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Hi all,
I need a low-bias library for high-resolution RNA footprinting, so that the footprint boundaries are clearly resolved. I am not talking about broad 3' or 5' bias, its rather "patchy" coverage that I'm trying to avoid.
So far I have read a few papers on this and the consensus seems to be that the ligations of single-stranded adapters to the RNA fragments using RNA ligases greatly distorts the library (both the 5' and 3' ligation reactions); while the polyadenylation approach followed by TnVN-primed RT, and cDNA circularization, is the best approach [1,2].
However, I have a library prep kit from ABI with a slightly different approach: apparently they hybridize ds adaptors with NNNN overhangs (a 3' NNNN overhang for the 5' adaptor and a 5' NNNN overhang for the 3' adaptor) and ligate with a "ligation enzyme mix". This is quite different I imagine, because it is more of a nick-repair reaction than a ssRNA ligation reaction (and I think that the adapters might be DNA not RNA). This technique was not analysed in these papers. Does anyone have some data on the evenness/patchyness of coverage with this library prep technique?
1 - Joshua Z Levin et. al. Nat Methods 2010
2 - Anitha D. Jayaprakash et al. Nuc Acids Res 2011
I need a low-bias library for high-resolution RNA footprinting, so that the footprint boundaries are clearly resolved. I am not talking about broad 3' or 5' bias, its rather "patchy" coverage that I'm trying to avoid.
So far I have read a few papers on this and the consensus seems to be that the ligations of single-stranded adapters to the RNA fragments using RNA ligases greatly distorts the library (both the 5' and 3' ligation reactions); while the polyadenylation approach followed by TnVN-primed RT, and cDNA circularization, is the best approach [1,2].
However, I have a library prep kit from ABI with a slightly different approach: apparently they hybridize ds adaptors with NNNN overhangs (a 3' NNNN overhang for the 5' adaptor and a 5' NNNN overhang for the 3' adaptor) and ligate with a "ligation enzyme mix". This is quite different I imagine, because it is more of a nick-repair reaction than a ssRNA ligation reaction (and I think that the adapters might be DNA not RNA). This technique was not analysed in these papers. Does anyone have some data on the evenness/patchyness of coverage with this library prep technique?
1 - Joshua Z Levin et. al. Nat Methods 2010
2 - Anitha D. Jayaprakash et al. Nuc Acids Res 2011