NEBNext rRNA Depletion Kit (Human/Mouse/Rat) Protocol (E6310)

Note: There is a formulation change to one of the components of the kit (NEB #E6318: NEBNext RNase H). The protocol and use of this component have not changed.


Starting Material:
10 ng–1 μg total RNA (DNA free) in a 12 μl total volume.

1.     Hybridize the Probes to the RNA
1.1.  Prepare a RNA/Probe master mix as follows: 
COMPONENT VOLUME
NEBNext rRNA Depletion Solution 1 μl
Probe Hybridization Buffer 2 μl
Total Volume 3 μl

1.2.  Add 3 μl of the above mix to 12 μl total RNA sample.
1.3.  Mix by pipetting up and down at least 10 times.
1.4.  Spin down briefly in a tabletop centrifuge, and immediately proceed to the next step.
1.5.  Place samples in a thermocycler with a heated lid set to approximately 105°C, and run the following program, which will take approximately 15–20 minutes to complete:

TEMP TIME
95°C 2 min
95-22°C 0.1°C/sec
22°C 5 min hold

1.6.  Spin down the samples in a tabletop centrifuge, and place on ice. Proceed immediately to the next step.

2.     RNase H Digestion
2.1.  On ice, prepare a master mix according to the following table, and mix by pipetting up and down at least 10 times; use immediately.

COMPONENT VOLUME
NEBNext RNase H 2 μl
RNase H Reaction Buffer 2 μl
Nuclease-free Water 1 μl
Total Volume 5 μl

2.2.  Add 5 μl of the above mix to the RNA sample from Step 1.6.
2.3. Mix by pipetting up and down at least 10 times.
2.4.  Spindown briefly in a table top centrifuge and immediately proceed to the next step.
2.5.  Place samples in a thermocycler (with lid at 40°C) and incubate at 37°C for 30 minutes.
2.6.  Spin down the samples in a tabletop centrifuge, and place on ice. Proceed immediately to the next step to prevent non-specific degradation of RNA.

3.     DNase I Digestion.
3.1.  On ice, prepare a DNase I Digestion Master Mix according to the following table, and mix by pipetting up and down at least 10 times; use immediately.

COMPONENT VOLUME
DNase I Reaction Buffer 5 μl
DNase I (RNase-free) 2.5 μl
Nuclease-free Water 22.5 μl
Total Volume 30 μl

3.2.  Add 30 μl of the above mix to the RNA sample from Step 2.6.
3.3.  Mix by pipetting up and down at least 10 times.
3.4.  Spin down briefly in a table top centrifuge and immediately proceed to the next step.
3.5.  Place samples in a thermocycler (with lid at 40°C) and incubate at 37°C for 30 minutes.
3.6.  Spin down the samples in a tabletop centrifuge, and place on ice. Proceed immediately to the next step.
4.     RNA Purification after rRNA Depletion Using Agencourt RNAClean XP
4.1.  Vortex RNAClean XP bead to resuspend.
4.2.  Add 110 μl (2.2X) resuspended RNAClean XP beads to the RNA Sample. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out. 
4.3.  Incubate samples on ice for 15 minutes.
4.4.  Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
4.5.  After 5 minutes (or when the solution is clear), carefully remove and discard the supernatent. Be careful not to disturb the beads that contain the RNA (Caution: do not discard the beads).
4.6.  Add 200 μl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the RNA.
4.7.  Repeat Step 4.6 once for a total of 2 washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
4.8.  Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open. 
Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.
4.9.  Remove the tube/plate from the magnetic stand. Elute the RNA from the beads by adding 8 μl of nuclease free water. 
4.10.Place the tube/plate on a magnetic stand. After 5 minutes (or when the solution is clear), transfer 6 μl to a new PCR tube.
4.11.Place the tube on ice and proceed with NGS library construction or other downstream application. Alternatively, the samples can be stored at -20°C.