The following are the steps for running a PCR free method for Cell3 Target library prep:
- Fragmentation/End repair/A-tailing (use 1 ul for tapestation to verify size)
- Ligation of adapters
- Bead clean-up
- Quantitation by qPCR (not Qubit and TapeStation)
When conducting a PCR free library prep, the QC at the end (following adapter ligation) will not give accurate Qubit readings (they will appear lower than what they should be) and the Tapestation will not show fragments in the right range. This is due to the ligation of Y-shaped adapters.
The best way to QC is using qPCR. If you want to confirm the average fragment size, you could use 1 ul of the fragmentation reaction (post incubation) and run that on the tapestation. The average fragment length + adapters (144bp) will give the final library average fragment length.