Ligate sequencing adapters to the RNA fragment ends
Add 8 µl of NEBNext Quick Ligation Reaction Buffer to your reverse-transcribed RNA (Tube 4).
Add 6 µl of RNA Ligation Adapter (RLA) to Tube 4.
Add 3 µl nuclease-free water to the tube.
Add 3 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 4 for 10 minutes at room temperature. This is the adapter ligation reaction (Tube 4).
Resuspend the Agencourt RNAClean XP beads by vortexing.
Add 16 µl of resuspended RNAClean XP beads to the adapter ligation reaction (Tube 4), and mix by pipetting up and down.
Put the tube in a Hula mixer, and leave to incubate for 5 minutes.
Take Tube 4 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 5 minutes 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 Wash Buffer (WSB) to Tube 4. Close the tube lid, and resuspend the beads by flicking the tube with your finger.
Return the tube to the magnetic rack, allow the beads to pellet, then pipette off and discard the buffer.
Place the tube in a magnetic rack and wait for 5 minutes 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.
Repeat the previous three steps:
Add 150 µl of Wash Buffer (WSB) to Tube 4. Close the tube lid, and resuspend the beads by flicking the tube with your finger.
Return the tube to the magnetic rack, allow the beads to pellet for 5 minutes, then pipette off and discard the buffer.
Take the tube off the magnetic rack and spin down briefly in a microfuge.
Place Tube 2 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 13 µl RNA Elution Buffer (REB) to the tube, and resuspend the beads by flicking the tube.
Incubate for 10 minutes at room temperature.
Place the tube in a magnetic rack and wait for 5 minutes for the beads to collect in a pellet near the magnet and the solution to become clear.
Pipette off the 13 µl of eluate from Tube 4 and transfer it to a clean 1.5 ml Eppendorf DNA LoBind tube (Tube 5). Discard Tube 4 with the pellet.
Remove 1 µl of eluate from Tube 5 and use the Qubit fluorometer DNA HS assay to quantify the amount of RNA in the sample.
