This caution sign signifies a step in the protocol that has two paths leading to the same end point but is dependent on a user variable, like the type of RNA input.
	This is a point where you can safely stop the protocol and store the samples prior to proceeding to the next step in the protocol.
	Colored bullets indicate the cap color of the reagent to be added.

Keep all of the buffers on ice, unless otherwise indicated.

RNA Sample Requirements

RNA Integrity

Assess the quality of the input RNA by running the RNA sample on an Agilent Bioanalyzer RNA 6000 Nano/Pico Chip to determine the RNA Integrity Number (RIN). Both intact and degraded RNA can be used in the depletion protocol. However, processing of samples for different downstream applications may be impacted by the RIN scores.

RNA Purity

The RNA sample should be free of salts (e.g., Mg2+, or guanidinium salts) or organics (e.g., phenol and ethanol). RNA must be free of DNA. gDNA is a common contaminant from RNA preps. It may be carried over from the interphase of organic extractions or when the silica matrix of solid phase RNA purification methods is overloaded. If the total RNA sample may contain gDNA contamination, treat the sample with DNase I to remove all traces of DNA (not provided in this kit). After treatment with DNase I the enzyme should be removed from the sample. Any residual activity of DNase I will degrade the single stranded DNA probes necessary for the ribosomal depletion.

Input Amount

10 ng–1 μg total RNA (DNA free) in a maximum of 11 µl of nuclease-free water, quantified by an RNA-specific dye-assisted fluorometric method (e.g., Qubit^®, RiboGreen^®), and quality checked by Bioanalyzer.

1.1. Probe Hybridization to RNA

1.1.1. Dilute 10 ng–1 μg of total RNA with Nuclease-free Water to a final volume of 11 μl in a PCR tube. Keep the RNA on ice.

1.1.2. Assemble the following RNA/Probe hybridization reaction on ice:

RNA/PROBE HYBRIDIZATION REACTION	VOLUME
Total RNA in Nuclease-free Water (10 ng–1 µg)	11 µl
(white) NEBNext v2 rRNA Depletion Solution	2 µl
(white) NEBNext Probe Hybridization Buffer	2 µl
Total Volume	15 µl

1.1.3. Mix thoroughly by gently pipetting up and down at least 10 times. Note: It is crucial to mix well at this step.

1.1.4. Briefly spin down the tube in a microcentrifuge to collect the liquid from the side of the tube.

1.1.5. Place tube in a pre-heated thermocycler and run the following program with the heated lid set to 105°C. This program will take approximately 15-20 minutes to complete.

1.1.6. Briefly spin down the tube in a microcentrifuge and place on ice. Proceed immediately to RNase H Digestion.

1.2. RNase H Digestion

1.2.1. Assemble the following RNase H digestion reaction on ice:

RNASE H DIGESTION REACTION	VOLUME
Hybridized RNA (Step 1.1.6)	15 µl
(white) RNase H Reaction Buffer	2 µl
(white) NEBNext Thermostable RNase H	2 µl
Nuclease-free Water	1 µl
Total Volume	20 µl

1.2.2. Mix thoroughly by pipetting up and down at least 10 times.

1.2.3. Briefly spin down the tube in a microcentrifuge.

1.2.4. Incubate the tube in a pre-heated thermocycler for 30 minutes at 50°C with the lid set to 55°C.

1.2.5. Briefly spin down the tube in a microcentrifuge and place on ice. Proceed immediately to DNase I Digestion.

1.3. DNase I Digestion

1.3.1. Assemble the following DNase I digestion reaction on ice:

1.3.2. Mix thoroughly by pipetting up and down at least 10 times.

1.3.3. Briefly spin down the tube in a microcentrifuge.

1.3.4. Incubate in a pre-heated thermocycler for 30 minutes at 37°C with the heated lid set to 40°C (or off).

1.3.5. Briefly spin down the tube in a microcentrifuge and place on ice. Proceed immediately to RNA Purification.

1.4 RNA Purification using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads

1.4.1. Vortex the Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads to resuspend.

1.4.2. Add 90 µl (1.8X) beads to the RNA sample from Step 1.3.5 and mix thoroughly by pipetting up and down at least 10 times.

1.4.3. Incubate for 15 minutes on ice to bind RNA to the beads.

1.4.4. Place the tube on a magnetic rack to separate the beads from the supernatant.

1. 4.5. After the solution is clear, carefully remove and discard the supernatant. Be careful not to disturb the beads which contain the RNA.

1.4.6. Add 200 µl of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for 30 seconds and then carefully remove and discard the supernatant. Be careful not to disturb the beads which contain the RNA.

1.4.7. Repeat Step 1.4.6 once for a total of two washes.

1.4.8. Completely remove residual ethanol and air dry the beads for up to 5 minutes while the tube is on the magnetic rack 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.

1.4.9. Remove the tube from the magnetic rack. Elute the RNA from the beads by adding 7 μl of Nuclease-free Water. Mix thoroughly by pipetting up and down at least 10 times and briefly spin the tube.

1.4.10. Incubate for 2 minutes at room temperature.

1.4.11. Place the tube on the magnetic rack until the solution is clear (~ 2 minutes).

1.4.12. Remove 5 µl of the supernatant containing RNA and transfer to a nuclease-free tube.

1.4.13. Place the tube on ice and proceed with RNA-Seq library construction or other downstream application. Alternatively, the sample can be stored at -80°C.