Protocol for use with NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (E6310, E6350)

Protocol Starting Material: 5 ng–1 µg total RNA (DNA free) in a 12 µl total volume. If the total RNA may contain gDNA contamination, treat the RNA sample with DNase I to remove all traces of DNA, then purify the treated RNA to remove DNase I.

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 or off) 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 or off) 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 NEBNext RNA Sample Purification Beads


4.1. Vortex RNA sample purification beads to resuspend.

4.2. Add 110 μl (2.2X) resuspended RNA sample purification 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 supernatant. 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 two 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 RNA 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.

Mix well by pipetting up and down 10 times. Incubate for at least 2 minutes. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

4.10. Place the tube/plate on the 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 sample can be stored at –80°C

Recommended: To make sure rRNA is efficiently depleted, design RT aqPCR primers for the sample species rRNA and primers for a housekeeping gene. Compare rRNA content before and after ribosomal depletion to assess the rRNA removal efficiency.

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