Keep these on ice:

• RNA Calibration Strand (RCS)
• RNA Adapter Mix (RMX)
• 10 mM dNTP solution
• NEBNext® Quick Ligation Reaction Buffer

Defrost these at room temperature:

• RT Adapter (RTA)
• Wash Buffer (WSB)
• RNA Running Buffer (RRB)
• Elution Buffer (ELB)

Once the contents of each tube have thawed, mix the contents by flicking the tube and vortex the RNA Running Buffer (RRB), and spin down briefly in a microfuge.

Keep the reagent tubes on ice when they are not in use.

Set up a thermal cycler with the program 50°C for 50 minutes, then 70°C for 10 minutes, then hold at 4°C. Alternatively, set two heat blocks at 50°C and 70°C.