Keep these on ice:

• RT Adapter (RTA)
• RNA Calibration Strand (RCS)
• RNA Ligation Adapter (RLA)
• 10 mM dNTP solution

Defrost these at room temperature:

• NEBNext Quick Ligation Reaction Buffer
• T4 DNA Ligase

Once the contents of each tube have thawed, spin down the tubes in a microfuge and mix the contents by pipetting up and down for 10 full-volume pipette mixes.

Defrost these at room temperature:

• Wash Buffer (WSB)
• RNA Elution Buffer (REB)

Vortex both tubes, then spin them down and place on ice.

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.