Transfer 3 µl of NEBNext Quick Ligation Reaction Buffer into a clean 0.2 ml PCR tube (Tube 1).

Add 8.5 µl RNA Calibration Strand (RCS) to Tube 1.

Add 1 µl RNaseOUT to the tube.

Add 1 µl RT Adapter (RTA) to the tube.

Add 1.5 µl T4 DNA Ligase to the tube.

Mix the contents of the tube by pipetting up and down. Spin down briefly in a microfuge.

Incubate Tube 1 for 10 minutes at room temperature. This is the adapter ligation reaction (Tube 1).

Take a clean 1.5 ml Eppendorf DNA LoBind tube (Tube 2), and add 9 µl nuclease-free water.

Add 2 µl of 10 mM dNTPs to Tube 2.

Add 8 µl first strand buffer from the SuperScript III kit to Tube 2.

Add 4 µl of 0.1 M DTT to Tube 2.

Mix the contents of the tube by flicking the tube with your finger. Spin down briefly in a microfuge.

Add the contents of the 1.5 ml tube (Tube 2) to the 0.2 ml PCR tube (Tube 1) containing the RT adapter ligated RNA.

Mix the contents of the tube by pipetting up and down. Spin down briefly in a microfuge.

Add 2 µl of SuperScript III reverse transcriptase to the 0.2 ml tube (Tube 1).

Mix the contents of the tube by pipetting up and down. Spin down briefly in a microfuge.

Incubate in heat blocks or a thermal cycler at 50°C for 50 minutes, then at 70°C for 10 minutes, then hold at 4°C for approximately 30-60 seconds.
Take the tube out from the thermal cycler or heat block, and spin down briefly in a microfuge.

Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube (Tube 3). Tube 3 is the reverse transcription reaction.

Resuspend the Agencourt RNAClean XP beads by vortexing.

Add 72 µl of resuspended RNAClean XP beads to the reverse transcription reaction (Tube 3), and mix by pipetting up and down.

Put the tube in a Hula mixer, and leave to incubate for 5 minutes.

While the RNA sample is incubating, transfer 140 µl of pure ethanol to a clean 1.5 ml Eppendorf DNA LoBind tube.

Add 50 µl nuclease-free water to the ethanol.

Mix the tube of diluted ethanol by vortexing. Spin down briefly in a microfuge.

Take Tube 3 with the reverse transcription reaction off the Hula mixer, and spin down in a microfuge.

Place the tube in a magnetic rack, and wait for the beads to collect in a pellet near the magnet, and the solution to become clear.

Keep the tube on the magnet to pipette off and discard the supernatant. Take care to not disturb the pellet.

Add 150 µl of the freshly-prepared ethanol to Tube 3, without taking the tube off the magnetic rack or disturbing the pellet.

Keeping the magnetic rack on the benchtop, rotate the bead-containing tube by 180°. Wait for the beads to migrate towards the magnet and form a pellet.

Rotate the tube 180° again (back to the starting position), and wait for the beads to pellet.

After this, pipette off and discard the ethanol.

Take the tube off the magnetic rack and spin down briefly in a microfuge.

Place the tube back in the magnetic rack, and wait for the beads to collect in a pellet near the magnet. There may be a small amount of residual liquid at the bottom of the tube. Pipette off and discard this liquid.

Remove the tube from the magnetic rack, add 20 µl nuclease-free water to the tube, and resuspend the beads by flicking the tube.

Incubate for 5 minutes at room temperature.

Place the tube back in the magnetic rack, and wait for the beads to collect in a pellet near the magnet.

Pipette off the 20 µl of eluate from Tube 3, and transfer it to a clean 1.5 ml Eppendorf DNA LoBind tube (Tube 4). Discard Tube 3 with the pellet and retain Tube 4 with the eluate.