TABLE 2.
Primer duplex oligonucleotides used to capture ddA +/− 3′ddG 3′-labeled input RNA
| Name | Sequence | Mix ratio | Notes | |
|---|---|---|---|---|
| Previously described +1 Y duplex primers | +T.v2.Cy5 | /5Cy5/GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | 4 | |
| +C.v2.Cy5 | /5Cy5/GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCC | 1 | Incomplete R2 sequence. Paired-end sequencing will be inhibited. PCR amplification can be inhibited. | |
| RNA.v2 | rGrArUrCrGrGrArArGrAmGmCmAmCmAmCmGmUmCmUmGmAmAmCmUmCmCmAmGmU/3SpC3/ | 5 | 2′-O-methyl ribonucleotides are immune to RNase H, can potentially interfere with cDNA migration during dPAGE. | |
| Updated universal primers for +1 Y duplex | +T.v3.IR | /5IRD800/GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTT | 4 | Use updated adapter sequence during read trimming in the analysis pipeline. |
| +C.v3.IR | /5IRD800/GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTC | 1 | ||
| RNA.v3 | rArGrArUrCrGrGrArArGrAmGmCmAmCmAmCmGmUmCmUmGmAmAmCmUmCmCmAmGmU/3SpC3/ | 5 | 2′-O-methyl ribonucleotides are immune to RNase H, can potentially interfere with cDNA migration during dPAGE. | |
| Current buffer 4B2 primer duplex | +T.v2.IR | /5IRD800/GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | 1 | Current recommendation for ddA-only labeling. |
| RNA.v2 | rGrArUrCrGrGrArArGrAmGmCmAmCmAmCmGmUmCmUmGmAmAmCmUmCmCmAmGmU/3SpC3/ | 1 | 2′-O-methyl ribonucleotides are immune to RNase H, can potentially interfere with cDNA migration during dPAGE. | |
| Updated RNase H sensitive (buffer 4B2.1 primer duplex)* | +T.v2.IR* | /5IRD800/GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | 1 | Current recommendation for ddA-only 3′ labeling. |
| RNA.v2.1* | rGrArUrCrGrGrArArGrAmGmCmAmCmAmCmGmUrCrUrGrArArCrUmCmCmAmGmUmCmAmC/3SpC3/ | 1 | RNA ribonucleotides introduced between 2′-O-methyl ribonucleotides to ensure primer duplex sensitivity to RNase H. |
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Mix ratio is the molar ratio used to combine oligonucleotides before annealing. The asterisk indicates our current recommendation for both OTTR v1.3 and v2 cDNA library synthesis.
