In both LUTHOR HD and LUTHOR HD Pool, library generation is initiated by oligo(dT) priming, where the primer contains a 12 nt Unique Molecular Identifier (UMI) and a partial Illumina-compatible linker sequence. However, in LUTHOR HD Pool, this primer also contains a 12 nt i1 sample-barcode.
This early barcoding allows the samples to be combined by pooling after reverse transcription. All subsequent steps of the protocol can thus be performed on pools containing up to 96 samples. Batch processing significantly shortens the overall library generation time and reduces technical variability between samples within one pool.
For best practice, we would recommend keeping kit components separate and avoid mixing/matching. Importantly, IVT and E4 are NOT interchangeable between kits. Additionally, some steps between protocols may differ e.g., LUTHOR HD Pool includes an additional incubation step during purification THOR reaction (Step 7; bold): 10 minutes at 37 °C, 5 minutes at 42 °C, 20 minutes at 65 °C, then cool to 25 °C and hold for 1 minute.