Are there any special changes to QuantSeq or CORALL protocols when preparing dual-indexed libraries?
When using QuantSeq with UDI V2 (Cat. No. 191 - 196) or CORALL RNA-Seq V2 (Cat. No. 171 - 176, 177 - 182, and 183 - 186), simply follow the respective User Guide. No special changes in the protocol are required. The same applies for the V1 of these kits: QuantSeq FWD with 12nt UDI kits (Cat. No. 113 - 115, and 129 -131) or CORALL with 12nt UDI kits (Cat. No. 117 – 119, 132 – 134, 158 - 163, and 171 - 186).
For dual indexing of standard QuantSeq FWD / REV (Cat. No. 015 and 016) and CORALL Total RNA-Seq (Cat. No. 095, 146 - 147) kits, which are supplied with 6 nt i7 indices, the i5 6 nt Dual Indexing Add-on Kits are most compatible (Cat. No. 047).
The i5 and i7 indices are added during the final library amplification. Please follow the detailed protocol for Endpoint PCR and Post-PCR Purification steps for dual-indexed library preparation in the Instruction Manual for the Lexogen i5 6 nt Dual Indexing Add-on Kits (047IM109). Differences to the protocol for amplification of single-indexed QuantSeq libraries are as follows:
The Dual PCR Mix (purple cap) must be used instead of the standard PCR Mix (yellow cap).
The total volume of the PCR is 35 μl.
The volume of Purification Beads (PB) to add for post-PCR purification is 35 μl (or 31.5 μl for libraries generated from low input or degraded / FFPE RNA samples).
The Dual PCR Mix is provided in the Lexogen i5 6 nt Dual Indexing Add-on Kits along with the i5 index primers. The i7 index primers and Enzyme Mix (E3) are provided in the respective library prep kit. The purification reagents for Post-PCR Purification are also provided in the standard library prep kits.
If need be, the Enzyme Mix (E), from the PCR Add-on Kit for Illumina (Cat. No. 020.96) can also be used for dual indexing endpoint PCR (it is interchangeable with E3 from the standard kits).
NOTE: Alternatively, the 12 nt UDI Add-on Kits (Cat. No. 198 - 203) can also be used together with the above-listed standard library prep kits. Please note however, that use of the UDI Add-on Kits will mean that the i7 indices and the PCR Mix (yellow cap) supplied in the standard kits should not be used (and are best discarded). Therefore, to avoid wastage or protocol confusion, we highly recommend the use of the UDI Bundle options of the QuantSeq and CORALL library prep kits if you wish to use UDIs for your library preprations. Please see this online FAQ for more info on the available UDI Library Prep Kit Bundles.