TITLE: Protocol for use with FFPE RNA, NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E6310) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760, E7765)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina contains the enzymes and buffers required to convert a broad range of input amounts of RNA into high quality directional (strand-specific) libraries for next-generation sequencing on the Illumina platform. The fast, user-friendly workflow has minimal hands-on time and is compatible with poly(A) mRNA enrichment and rRNA depletion methods.</div></div>

[STEPS]
?. [Probe Hybridization to RNA]
Add  of the above mix to  total RNA (from Step 1), resulting in a total volume of
3 µl
12 µl
15 µl
?. [Probe Hybridization to RNA]
Mix by pipetting up and down at least 10 times.
?. [Probe Hybridization to RNA]
Spin down briefly in a microcentrifuge.
?. [Probe Hybridization to RNA]
Place samples in a thermocycler with a heated lid set to approximately , and run the following program, which will take approximately 15–20 minutes to complete:  AB1TemperatureTime295°C2 min3Ramp down to 22°C0.1°C/sec4Hold at 22°C5 minutes
?. [Probe Hybridization to RNA]
Spin down the samples in a microcentrifuge, and place on ice. Proceed immediately to RNase H Digestion Step.
?. [RNase H Digestion]
Assemble the RNAse H master mix on ice as follows.   AB1RNase H Master MixVolume2NEBNext RNase H2 μl3RNase H&nbsp;Reaction Buffer2 μl4Nuclease-free Water1 μl5Total Volume5 μl
?. [RNase H Digestion]
Mix thoroughly by pipetting up and down 10 times.
?. [RNase H Digestion]
Briefly spin down the samples in a microcentrifuge.
?. [RNase H Digestion]
Add  of the RNase H master mix to the RNA sample from Step 7, resulting in a total volume of
3 µl
20 µl
?. [RNase H Digestion]
Incubate the sample in a thermocycler for  at with the lid set to  (or on).
?. [RNase H Digestion]
Briefly spin down the samples in a microcentrifuge, and place on ice. Proceed immediately to DNase I Digestion to prevent non-specific degradation of RNA.
?. [DNase I Digestion]
Assemble the DNase I master mix on ice in a nuclease-free tube.  AB1DNase I Master MixVolume2DNase I Reaction Buffer5 μl3DNase I (RNase-free)2.5 μl4Nuclease-free Water22.5 μl5Total Volume30 μl
?. [DNase I Digestion]
Briefly spin down the sample in a microcentrifuge.
?. [DNase I Digestion]
Add  of DNase I master mix to  RNA sample from Step 14, resulting in a total volume of
30 µl
20 µl
50 µl
?. [DNase I Digestion]
Mix thoroughly by pipetting up and down 10 times.
?. [DNase I Digestion]
Incubate the sample in a thermocycler for  at  with the heated lid set to  (or on).
?. [DNase I Digestion]
Briefly spin down the sample in a microcentrifuge, and place on ice. Proceed immediately to RNA Purification.
?. [RNA Purification after rRNA Depletion Using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads]
Vortex Agencourt RNAClean XP Beads or RNA Sample Purification Beads to resuspend.
?. [RNA Purification after rRNA Depletion Using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads]
Add  (2.2X) beads to the RNA sample from Step 21 and mix thoroughly by pipetting up and down at least 10 times.
110 µl
?. [RNA Purification after rRNA Depletion Using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads]
Incubate the sample for  on ice to bind RNA to the beads.
?. [RNA Purification after rRNA Depletion Using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads]
Place the tube on a magnetic rack to separate beads from the supernatant. After the solution is clear, carefully remove and discard the supernatant. Be careful not to disturb the beads, which contain RNA.
?. [RNA Purification after rRNA Depletion Using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads]
Repeat Step 26 once for a total of 2 washing steps.
[Repeat Step]
?. [RNA Purification after rRNA Depletion Using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads]
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.
?. [RNA Purification after rRNA Depletion Using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads]
Remove the tube from the magnet. Elute the RNA from the beads by adding Nuclease-free Water. Mix well by pipetting up and down at least 10 times and briefly spin the tube.
7 µl
?. [RNA Purification after rRNA Depletion Using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads]
Incubate for  at room temperature. Place the tube in the magnet until the solution is clear (~).
?. [RNA Purification after rRNA Depletion Using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads]
Remove  of the supernatant containing RNA and transfer to a nuclease-free tube.
5 µl
?. [RNA Purification after rRNA Depletion Using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads]
Place the sample on ice and proceed to Priming of Highly Degraded RNA.
?. [Priming of Highly Degraded RNA (FFPE) Which has a RIN ≤ 2 and Does not Require Fragmentation]
Assemble the following fragmentation and priming reaction on ice:  AB1Priming ReactionVolume2rRNA Depleted Sample (Step 32)5 µl3Random Primers1 µl4Total Volume6 µl
?. [Priming of Highly Degraded RNA (FFPE) Which has a RIN ≤ 2 and Does not Require Fragmentation]
Mix thoroughly by pipetting up and down 10 times.
?. [Probe Hybridization to RNA]
Prepare a RNA/Probe master mix as follows:  AB1RNA Probe Master MixVolume2NEBNext rRNA Depletion Solution1 μl3Probe Hybridization Buffer2&nbsp;μl4Total Volume&nbsp; &nbsp; &nbsp;&nbsp;3&nbsp;μl
?. [Probe Hybridization to RNA]
Dilute the total RNA with Nuclease-free Water to a final volume of  in a PCR tube. Keep the RNA on ice.
12 µl
?. [RNase H Digestion]
Mix thoroughly by pipetting up and down at least 10 times.
?. [DNase I Digestion]
Mix thoroughly by pipetting up and down 10 times.
?. [RNA Purification after rRNA Depletion Using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads]
Add of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the RNA.
200 µl
?. [Priming of Highly Degraded RNA (FFPE) Which has a RIN ≤ 2 and Does not Require Fragmentation]
Incubate the sample in a preheated thermocycler as follows:  at , with heated lid set at Hold at
?. [Priming of Highly Degraded RNA (FFPE) Which has a RIN ≤ 2 and Does not Require Fragmentation]
Transfer the tube directly to ice and proceed to First Strand cDNA Synthesis.
?. [First Strand cDNA Synthesis]
Assemble the first strand synthesis reaction on ice by adding the following components to the fragmented and primed RNA from Step 37:  AB1First Strand Synthesis ReactionVolume2Primed RNA (Step 37)6 µl3NEBNext First Strand Synthesis Reaction Buffer4 µl4NEBNext Strand Specificity Reagent8 µl5NEBNext First Strand Synthesis Enzyme Mix2 µl6Total Volume20 µl
?. [First Strand cDNA Synthesis]
Mix thoroughly by pipetting up and down 10 times.
?. [First Strand cDNA Synthesis]
[ ! ] Incubate the sample in a preheated thermocycler with the heated lid set at ≥  as follows:Note: If you are following recommendations in Appendix A (Chapter 6), for libraries with longer inserts (> 200 bases), increase the incubation at 42°C from 15 minutes to 50 minutes at Step 2 below.Step 1:  at Step 2:  at Step 3:  at Step 4: Hold at
?. [First Strand cDNA Synthesis]
Proceed directly to Second Strand cDNA Synthesis.
?. [Second Strand cDNA Synthesis]
Assemble the second strand cDNA synthesis reaction on ice by adding the following components into the first strand synthesis product from Step 40.  AB1Second&nbsp;Strand Synthesis ReactionVolume2First&nbsp;Strand Synthesis Product (Step 40)20 µl3NEBNext Second Strand Synthesis Reaction Buffer with dUTP (10X)8 µl4NEBNext Second Strand Synthesis Enzyme Mix4 µl5Nuclease-free Water48 µl6Total Volume80 µl
?. [Second Strand cDNA Synthesis]
Keeping the tube on ice, mix thoroughly by pipetting up and down at least 10 times.
?. [Second Strand cDNA Synthesis]
Incubate in a thermocycler for  at  with the heated lid set at ≤  (or off).
?. [Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads]
Vortex SPRIselect Beads or NEBNext Sample Purification Beads to resuspend.
?. [Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads]
Add (1.8X) of resuspended beads to the second strand synthesis reaction (~). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
144 µl
80 µl
?. [Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads]
Incubate for  at room temperature.
?. [Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads]
Briefly spin the tube in a microcentrifuge to collect any sample on the sides of the tube. Place the tube on a magnet to separate beads from the supernatant. After the solution is clear, carefully remove and discard the supernatant. Be careful not to disturb the beads, which contain DNA. Caution: Do not discard beads.
?. [Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads]
Add  of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for , and then carefully remove and discard the supernatant.
200 µl
?. [Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads]
Repeat Step 49 once for a total of 2 washing steps.
?. [Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads]
Air dry the beads for up to 5 minutes while the tube is on the magnetic rack with 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.
?. [Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads]
Remove the tube from the magnetic rack. Elute the DNA from the beads by adding  0.1X TE Buffer (provided) to the beads. Mix well on a vortex mixer or by pipetting up and down at least 10 times. Quickly spin the tube and incubate for  at room temperature. Place the tube on the magnetic rack until the solution is clear.
53 µl
?. [Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads]
Remove  of the supernatant and transfer to a clean nuclease-free PCR tube.
50 µl
If you need to stop at this point in the protocol samples can be stored at –20°C.
?. [End Prep of cDNA Library]
Assemble the end prep reaction on ice by adding the following components to the second strand synthesis product from Step 53.  AB1End Prep ReactionVolume2Second Strand Synthesis Product (Step 52)50 µl3NEBNext Ultra II End Prep Reaction Buffer7 µl4NEBNext Ultra II End Prep Enzyme Mix3 µl5Total Volume60 µlIf a master mix is made, add  of master mix to of cDNA for the End Prep reaction.
10 µl
50 µl
?. [End Prep of cDNA Library]
Set a 100 μl or 200 μl pipette to 50 μl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
It is important to mix well. The presence of a small amount of bubbles will not interfere with performance.
?. [End Prep of cDNA Library]
Incubate the sample in a thermocycler with the heated lid set at ≥  as follows. at  at Hold at
?. [End Prep of cDNA Library]
Proceed immediately to Adaptor Ligation.
?. [Adaptor Ligation]
[ ! ] Dilute the NEBNext Adaptor* prior to setting up the ligation reaction in ice-cold Adaptor Dilution Buffer and keep the adaptor on ice.  AB1Total RNA InputDilution Required2100 ng–10 ng25–fold dilution in Adaptor Dilution Buffer*The NEBNext adaptor is provided in NEBNext oligos kit. NEB has several oligo kit options, which are supplied separately from the library prep kit.
?. [Adaptor Ligation]
Assemble the ligation reaction on ice by adding the following components, in the order given, to the end prep reaction product from Step 5.  AB1Ligation ReactionVolume2End Prepped DNA (Step 57)60 µl3Diluted Adaptor (Step 57)2.5 µl4NEBNext Ligation Enhancer1 µl5NEBNext Ultra II Ligation Master Mix30 µl6Total Volume93.5 µl
The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours @ 4°C. We do not recommend premixing the Ligation Master Mix, Ligation Enhancer and adaptor prior to use in the Adaptor Ligation Step.
?. [Adaptor Ligation]
Set a 100 μl or 200 μl pipette to 80 μl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.Caution: The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance.
?. [Adaptor Ligation]
Incubate  at  in a thermocycler.
?. [Adaptor Ligation]
Add  (blue) USER Enzyme to the ligation mixture from Step 60, resulting in total volume of
3 µl
96 µl
?. [Adaptor Ligation]
Mix well and incubate at for  with the heated lid set to ≥
?. [Adaptor Ligation]
Proceed immediately to Purification of the Ligation Reaction.
?. [Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads]
[ ! ] Note: If you are selecting for libraries with larger insert size (> 200 nt) follow the size selection recommendations in Appendix A, Chapter 6. Add  (0.9X) resuspended SPRIselect Beads or NEBNext Sample Purification Beads and mix well on a vortex mixer or by pipetting up and down at least 10 times.
87 µl
?. [Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads]
Incubate for  at room temperature.
?. [Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads]
Quickly spin the tube in a microcentrifuge and place the tube on an appropriate magnetic rack to separate beads from the supernatant. After the solution is clear (~), discard the supernatant that contains unwanted fragments.Caution: Do not discard beads.
?. [Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads]
Add  of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for , and then carefully remove and discard the supernatant.
200 µl
?. [Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads]
Repeat Step 68 once for a total of 2 washing steps.
[Repeat Step]
?. [Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads]
Briefly spin the tube, and put the tube back in the magnetic rack.
?. [Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads]
Completely remove the residual ethanol, and air dry beads until the beads are dry 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 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.
?. [Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads]
Remove the tube from the magnetic rack. Elute DNA target from the beads by adding  0.1X TE (provided) to the beads. Mix well on a vortex mixer or by pipetting up and down. Quickly spin the tube and incubate for  at room temperature. Put the tube in the magnet until the solution is clear.
17 µl
?. [Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads]
Without disturbing the bead pellet, transfer  of the supernatant to a clean PCR tube and proceed to PCR enrichment.
15 µl
If you need to stop at this point in the protocol samples can be stored at –20°C.
?. [PCR Enrichment of Adaptor Ligated DNA]
[ ! ] Check and verify that the concentration of your oligos is 10 μM on the label.[ ! ] Use Option A for any NEBNext oligos kit where index primers are supplied in tubes. These kits have the forward and reverse primers supplied in separate tubes.Use Option B for any NEBNext oligos kit where index primers are supplied in a 96-well plate format. These kits have the forward and reverse (i7 and i5) primers combined.
?. [PCR Enrichment of Adaptor Ligated DNA]
Set up the PCR reaction as described below based on the type of oligos (PCR primers) used.Option A: Forward and Reverse Primers Separate  AB1ComponentVolume Per One Library2Adaptor Ligated DNA (Step 73)15 µl3NEBNext Ultra II Q5 Master Mix25 µl4Universal PCR Primer/i5 Primer*,**5 µl5Index (X) Primer/i7 Primer*,**5 µl6Total Volume50 µlOption B: Forward and Reverse Primers Combined  AB1ComponentVolume Per One Library2Adaptor ligated DNA (Step 73)15 µl3NEBNext Ultra II Q5 Master Mix25 µl4Index (X)/i7 Primer Mix*10 µl5Total Volume50 µl* NEBNext Oligos must be purchased separately from the library prep kit. Refer to the corresponding NEBNext Oligo kit manual for determining valid barcode combinations.** Use only one i7 primer/ index primer per sample. Use only one i5 primer (or the universal primer for single index kits) per sample
?. [PCR Enrichment of Adaptor Ligated DNA]
Mix well by gently pipetting up and down 10 times. Quickly spin the tube in a microcentrifuge.
?. [PCR Enrichment of Adaptor Ligated DNA]
Place the tube on a thermocycler with the heated lid set to  and perform PCR amplification using the following PCR cycling conditions (refer to Table 77.A and Table 77.B):Table 77.A:  ABCD1Cycle StepTempTimeCycles2Initial&nbsp;Denaturation98°C30 seconds13Denaturation98°C10 seconds12–16*,**4Annealing/Extension65°C75 seconds5Final Extension65°C5 minutes16Hold4°C∞&nbsp;* The number of PCR cycles should be adjusted based on RNA input.** It is important to limit the number of PCR cycles to avoid overamplification. If overamplification occurs, a second peak ~ 1,000 bp will appear on the Bioanalyzer trace (see Figure 7.2 in manual).Table 77.B: Recommended PCR cycles based on input amount:  AB1FFPE RNA InputRecommended PCR Cycles2100 ng12–13310 ng15–16
?. [Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads]
Vortex SPRIselect Beads or NEBNext Sample Purification Beads to resuspend.
?. [Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads]
Add  (0.9X) of resuspended beads to the PCR reaction (~). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
45 µl
50 µl
?. [Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads]
Incubate for  at room temperature.
?. [Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads]
Quickly spin the tube in a microcentrifuge and place the tube on an appropriate magnetic rack to separate beads from the supernatant. After the solution is clear (~), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets. Caution: Do not discard beads.
?. [Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads]
Add  of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for , and then carefully remove and discard the supernatant.
200 µl
?. [Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads]
Repeat Step 82 once for a total of 2 washing steps.
[Repeat Step]
?. [Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads]
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 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.
?. [Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads]
Remove the tube from the magnetic rack. Elute the DNA target from the beads by adding  0.1X TE (provided) to the beads. Mix well on a vortex mixer or by pipetting up and down ten times. Quickly spin the tube in a microcentrifuge and incubate for  at room temperature. Place the tube in the magnetic rack until the solution is clear.
23 µl
?. [Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads]
Transfer  of the supernatant to a clean PCR tube, and store at –20°C.
20 µl
?. [Assess Library Quality on an Agilent Bioanalyzer DNA Chip]
Run  library on a DNA High Sensitivity Chip. A dilution may be necessary for running on a Bioanalyzer High Sensitivity DNA Chip
1 µl
?. [Assess Library Quality on an Agilent Bioanalyzer DNA Chip]
Check that the electropherogram shows a narrow distribution with a peak size approximately 300 bp.
Note: If a peak at ~ 80 bp (primers) or 128 bp (adaptor-dimer) is visible in the bioanalyzer traces, bring up the sample volume (from Step 86) to 50 μl with 0.1X TE buffer and repeat the SPRIselect Bead or NEBNext Sample Purification Bead Cleanup Step (Section "Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads").
?. [Assess Library Quality on an Agilent Bioanalyzer DNA Chip]
Figure 89: Example of RNA library size distribution on a Bioanalyzer.
?. [Priming of Highly Degraded RNA (FFPE) Which has a RIN ≤ 2 and Does not Require Fragmentation]
Briefly spin down the samples in a microcentrifuge.