The most critical steps of QuantSeq library generation for the FWD and REV 3' mRNA-Seq protocols are during first strand cDNA synthesis. Our detailed handling guidelines ensure the sample temperature is maintained at the optimal range of 42 degrees Celcius. This ensures specific hybridization of the oligo(dT) primer at transcript poly(A) tails and prevents internal priming (mishybridization).
...
Prepare your RNA in 5 μl volumes and bring to room temperature for 2-5 minutes before adding the FS1 buffer.
Ensure FS1 is also fully equilibrated to room temperature. ATTENTION! Do not place RNA / FS1 samples back on ice and proceed immediately to denaturing after FS1 is added.
At step 3, pre-warm the FS2 / E1 mastermix for 2 – 3 minutes at 42 °C while the RNA / FS1 samples are denaturing for 3 minutes at 85 °C – Do not cool the mastermix on ice!
After the RNA / FS1 samples have cooled to 42 °C, spin these down briefly and then immediately return to the thermocycler and hold at 42 °C.
Add the pre-warmed FS2 / E1 mastermix to the RNA / FS1 samples on the thermocycler at 42 °C (step 4) and mix properly. Any drop in temperature at this point can cause mishybridization! Seal the plate or tubes and begin the 42 °C incubation.
After the 42 °C incubation is complete, proceed immediately to RNA Removal. Do not cool the samples on ice, or store below room temperature at this point!
NOTE: Spin down the samples at room temperature before and after adding the FS2 / E1 mastermix.
...
After the 42 °C incubation is complete, spin down the plate/tubes briefly and place at room temperature (or place back on the thermocycler at 42 °C)
Remove the sealing foil / tube caps, and immediately add the RNA Removal Solution (RS, thawed at room temperature) to the samples, mix well.
Briefly spin down the plate / tubes at room temperature, then place on the thermocycler to commence the 10 minute incubation at 95 °C (step 6).
NOTE: To minimize temperature drops at this point, the reactions can also be kept at 42 °C while the RNA Removal Solution (RS) is added.
...