What are the most critical steps in the library generation?

First Strand cDNA Synthesis:

• Do not cool the mastermix containing E1 (step 3).

• Have the RNA / Custom Targeted Primer or RNA / FS/ DTT / dT samples at 42 – 50 °C when adding the E1-containing mastermix (step 4) to avoid mishybridization.

• Mix properly by pipetting.

• Do not forget to shortly spin down the samples at room temperature before and after adding the E1-containing mastermix.