Protocol for use with NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (E6310) and NEBNext® Ultra II RNA Library Prep Kit for Illumina® (E7770, E7775)

Symbols
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.
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. 
Colored bullets indicate the cap color of the reagent to be added

The protocol has been optimized using high quality Universal Human Reference Total RNA.

RNA Sample Recommendations
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). RNA with different RIN values require different fragmentation times or no fragmentation at all.

For intact (RIN > 7) or partially degraded RNA samples (RIN = 2 to 7) follow the library preparation protocol in Chapter 2 (current chapter). See Table 2.5.1. for the recommended fragmentation times, based on RIN.

For highly degraded samples (RIN = 1 to 2) (e.g. FFPE), which do not require fragmentation, follow the library preparation protocol in Chapter 3.

RNA Purity:
Treat the RNA sample with DNase I to remove all traces of DNA. Remove DNase I after treatment. The RNA sample should be free of salts (e.g., Mg2+, or guanidinium salts), divalent cation chelating agents (e.g. EDTA, EGTA, citrate), or organics (e.g., phenol and ethanol).

Input Amount Requirement
5 ng – 1 µg total RNA (DNA-free) in up to 12 μl of Nuclease-free Water, quantified by Qubit Fluorometer and quality checked by Bioanalyzer.

The protocol is optimized for approximately 200 nt RNA inserts. To generate libraries with longer RNA insert sizes, refer to Appendix A (Chapter 6) for recommended fragmentation times and size selection conditions.

2.1. Probe Hybridization to RNA

2.1.1. Dilute the total RNA with Nuclease-free Water to a final volume of 12 μl in a PCR tube. Keep the RNA on ice.

2.1.2. Prepare a RNA/Probe master mix as follows:

RNA/PROBE MASTER MIX VOLUME
NEBNext rRNA Depletion Solution 1 µl
Probe Hybridization Buffer
2 µl
Total Volume
3 µl


2.1.3. Add 3 µl of the above mix to 12 µl total RNA (from Step 2.1.1), resulting in a total volume of 15 µl.

2.1.4. Mix thoroughly by pipetting up and down several times.

2.1.5. Briefly spin down the sample in a microcentrifuge.

2.1.6. Place samples in a thermal cycler, and run the following program with the heated lid set at 105°C. This will take approximately 15-20 minutes to complete.
2 minutes at 95°C
Ramp down to 22°C at 0.1°C/sec
5 minutes hold at 22°C

2.1.7. Briefly spin down the sample in a microcentrifuge, and place on ice. Proceed immediately to RNase H Digestion Step.

2.2. RNase H Digestion

2.2.1. Assemble the RNAse H master mix on ice as follows.

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


2.2.2. Mix thoroughly by pipetting up and down several times.

2.2.3. Briefly spin down the samples in a microcentrifuge.

2.2.4. Add 5 µl of the RNase H master mix to the RNA sample from Step 2.1.7, resulting in a total volume of 20 µl.

2.2.5. Mix thoroughly by pipetting up and down several times.

2.2.6. Incubate the sample in a thermal cycler for 30 minutes at 37°C with the lid set to 40°C.

2.2.7. 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.

2.3. DNase I Digestion

2.3.1. Assemble the DNase I master mix on ice in a nuclease-free tube.

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


2.3.2. Mix thoroughly by pipetting up and down several times.

2.3.3. Briefly spin down the sample in a microcentrifuge.

2.3.4. Add 30 μl of DNase I master mix to 20 μl RNA sample from Step 2.2.7, resulting in a total volume of 50 μl.

2.3.5. Mix thoroughly by pipetting up and down several times.

2.3.6. Incubate the sample in a thermal cycler for 30 minutes at 37°C with the heated lid set to 40°C.

2.3.7. Briefly spin down the sample in a microcentrifuge, and place on ice. Proceed immediately to RNA Purification.

2.4 RNA Purification Using Agencourt RNAClean® XP Beads or NEBNext RNA Sample Purification Beads

2.4.1. Vortex the RNAClean XP or RNA Sample Purification Beads to resuspend.

2.4.2. Add 110 µl (2.2X) beads to the RNA sample from Step 2.3.7 and mix thoroughly by pipetting up and down at least 10 times.

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

2.4.4. 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.

2.4.5. 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

2.4.6. Repeat Step 2.4.5 once for a total of 2 washing steps.

2.4.7. Completely remove residual ethanol, and air dry the beads for ~2-5 minutes while the tube is on the magnetic rack with the lid open.

Caution: Do not overdry the beads. This may result in lower recovery of RNA.  

2.4.8. Remove the tube from the magnet. Elute the RNA from the beads by adding 7 µl Nuclease-free Water. Mix well by pipetting up and down several times and briefly spin the tube.

2.4.9. Incubate for 2 minutes at room temperature. Place the tube in the magnet until the solution is clear (~2 minutes).

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

2.4.11. Place the sample on ice and proceed to RNA Fragmentation and Priming.

2.5. RNA Fragmentation and Priming

RNA fragmentation is only required for intact or partially degraded RNA. Recommended fragmentation times can be found in Table 2.5.1. 

2.5.1. Assemble the following fragmentation and priming reaction on ice

FRAGMENTATION AND PRIMING REACTION
VOLUME
Ribosomal RNA Depleted Sample (Step 2.4.11)   5 µl
(lilac) NEBNext First Strand Synthesis Reaction Buffer
4 µl
(lilac) Random Primers 
1 µl
Total Volume
10 µl


2.5.2. Mix thoroughly by pipetting up and down several times.

2.5.3. Place the sample on a thermal cycler and incubate the sample at 94°C following the recommendations in Table 2.5.1 below for fragment sizes ~200 nt. 

Table 2.5.1. Suggested fragmentation times based on RIN value of RNA input.

RNA TYPE RIN FRAG TIME
Intact RNA > 7   15 min. at 94°C
Partially Degraded RNA  2–6 7–8 min. at 94°C


Note: Refer to Appendix A for fragmentation conditions if you are preparing libraries with large inserts (> 200 bp). Conditions in Appendix A only apply for intact RNA.

2.5.4. Immediately transfer the tube to ice and proceed to First Strand cDNA Synthesis.

2.6. First Strand cDNA Synthesis

2.6.1. Assemble the first strand synthesis reaction on ice by adding the following components to the fragmented and primed RNA from Step 2.5.4: 

FIRST STRAND SYNTHESIS REACTION VOLUME
Fragmented and Primed RNA (Step 2.5.4)    10 µl
Nuclease-free Water
8 µl
(lilac) NEBNext First Strand Synthesis Enzyme Mix
2 µl
Total Volume
20 µl


2.6.2. Mix thoroughly by pipetting up and down several times.

2.6.3.  Incubate the sample in a preheated thermal cycler with the heated lid set at ≥ 80°C as follows:

Note: If you are following recommendations in Appendix A, for longer RNA fragments, increase the incubation at 42°C from 15 minutes to 50 minutes at Step 2.
Step 1: 10 minutes at 25°C
Step 2: 15 minutes at 42°C
Step 3: 15 minutes at 70°C
Step 4: Hold at 4°C

2.6.4. Proceed directly to Second Strand cDNA Synthesis.

2.7. Second Strand cDNA Synthesis

2.7.1. Assemble the second strand cDNA synthesis reaction on ice by adding the following components into the first strand synthesis product from Step 2.6.4.

SECOND STRAND SYNTHESIS REACTION VOLUME
First Strand Synthesis Product (Step 2.6.4) 20 µl
(orange) NEBNext Second Strand Synthesis Reaction Buffer (10X)
8 µl
(orange) NEBNext Second Strand Synthesis Enzyme Mix 
4 µl
Nuclease-free Water
48 µl
Total Volume
80 µl


2.7.2. Keeping the tube on ice, mix thoroughly by pipetting up and down several times. 

2.7.3. Incubate in a thermal cycler for 1 hour at 16°C with the heated lid set at ≤ 40°C.

2.8. Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads

2.8.1. Vortex SPRIselect beads or NEBNext Sample Purification Beads to resuspend.

2.8.2. Add 144 μl (1.8X) of resuspended beads to the second strand synthesis reaction (~80 μl). Mix well on a vortex mixer or by pipetting up and down at least 10 times.

2.8.3. Incubate for 5 minutes at room temperature.

2.8.4. 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.

2.8.5. 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.

2.8.6. Repeat Step 2.8.5 once for a total of 2 washing steps.  

2.8.7. Air dry the beads for 5 minutes while the tube is on the magnetic rack with lid open.

Caution: Do not overdry the beads. This may result in lower recovery of DNA.   

2.8.8. Remove the tube from the magnetic rack. Elute the DNA from the beads by adding 53 μl 0.1X TE Buffer (provided) to the beads. Mix well on a vortex mixer or by pipetting up and down several times. Quickly spin the tube and incubate for 2 minutes at room temperature. Place the tube on the magnetic rack until the solution is clear. 

2.8.9. Remove 50 µl of the supernatant and transfer to a clean nuclease-free PCR tube.

 If you need to stop at this point in the protocol, samples can be stored at –20°C.

2.9. End Prep of cDNA Library

2.9.1. Assemble the end prep reaction on ice by adding the following components to the second strand synthesis product from Step 2.8.9.

END PREP REACTION VOLUME
Second Strand Synthesis Product (Step 2.8.9) 50 µl
(green) NEBNext Ultra II End Prep Reaction Buffer 
7 µl
(green) NEBNext Ultra lI End Prep Enzyme Mix 
3 µl
Total Volume
60 µl


2.9.2. 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.

Note: It is important to mix well. The presence of a small amount of bubbles will not interfere with performance.

2.9.3. Incubate the sample in a thermal cycler with the heated lid set at ≥ 75°C as follows.
30 minutes at 20°C
30 minutes at 65°C
Hold at 4°C.

2.9.4. Proceed immediately to Adaptor Ligation.

2.10. Adaptor Ligation

2.10.1. Dilute the (red) NEBNext Adaptor* prior to setting up the ligation reaction in ice-cold Adaptor Dilution Buffer and keep the adaptor on ice. 

TOTAL RNA INPUT DILUTION REQUIRED
1,000 ng–101 ng 5-fold dilution in Adaptor Dilution Buffer
100 ng–10 ng 
25-fold dilution in Adaptor Dilution Buffer
5 ng 
200-fold dilution in Adaptor Dilution Buffer


*The adaptor is provided in NEBNext Singleplex (NEB #E7350) or NEBNext Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E6609 or #E7600) Oligos for Illumina.

2.10.2. Assemble the ligation reaction on ice by adding the following components, in the order given, to the end prep reaction product from Step 2.9.4.

LIGATION REACTION VOLUME
End Prepped DNA (Step 2.9.4)  60 µl
Diluted Adaptor (Step 2.10.1)  
2.5 µl
(red) NEBNext Ligation Enhancer
1 µl
(red) NEBNext Ultra II Ligation Master Mix
30 µl
Total Volume
93.5 µl


Note: 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.

2.10.3. 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.

2.10.4. Incubate 15 minutes at 20°C in a thermal cycler.

2.10.5. Add 3 μl (red) USER Enzyme to the ligation mixture from Step 2.10.4, resulting in total volume of 96.5 μl. 

  Note: Steps 2.10.5 and 2.10.6 are only required for use with NEBNext Adaptors. USER enzyme can be found in the NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E6609 or #E7600) Oligos for Illumina.

2.10.6. Mix well and incubate at 37°C for 15 minutes with the heated lid set to ≥ 45°C.

2.10.7. Proceed immediately to Purification of the Ligation Reaction.

2.11.  Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads


If you are selecting for larger size fragments (> 200 nt) follow the size selection recommendations in Appendix A, Chapter 6.

2.11.1. Add 87 μl (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.

2.11.2. Incubate for 5 minutes at room temperature.

2.11.3. 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 (~ 5 minutes), discard the supernatant that contains unwanted fragments. Caution: do not discard the beads.

2.11.4. 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.

2.11.5. Repeat Step 2.11.4 once for a total of 2 washing steps.  

2.11.6. Briefly spin the tube, and put the tube back in the magnetic rack.

2.11.7. Completely remove the residual ethanol, and air dry beads until the beads are dry for 5 minutes while the tube is on the magnetic rack with the lid open.

Caution: Do not overdry the beads. This may result in lower recovery of DNA.


2.11.8. Remove the tube from the magnetic rack. Elute DNA target from the beads by adding 17 μl 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 2 minutes at room temperature. Put the tube in the magnet until the solution is clear.

2.11.9. Without disturbing the bead pellet, transfer 15 μl of the supernatant to a clean PCR tube and proceed to PCR enrichment.

 If you need to stop at this point in the protocol, samples can be stored at –20°C.

2.12.    PCR Enrichment of Adaptor Ligated DNA
 
  Check and verify that the concentration of your oligos is 10 μM on the label.

  Follow Section 2.12.1Aif you are using the following oligos (10 µM):
    NEBNext Singleplex Oligos for Illumina (NEB #E7350)
    NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335)
    NEBNext Multiplex Oligos for Illumina (Set 2, NEB #E7500)
    NEBNext Multiplex Oligos for Illumina (Set 3, NEB #E7710)
    NEBNext Multiplex Oligos for Illumina (Set 4, NEB #E7730)
    NEBNext Multiplex Oligos for Illumina (Dual Index Primers, NEB #E7600)

  Follow Section 2.12.1B if you are using NEBNext Multiplex Oligos for Illumina (96 Index Primers, NEB #E6609).

2.12.1.    Set up the PCR reaction as described below based on the type of oligos (PCR primers) used.

2.12.1A    Forward and Reverse Primers Separate

COMPONENT VOLUME PER ONE LIBRARY
Adaptor Ligated DNA (Step 2.11.9)   15 µl
(blue) NEBNext Ultra II Q5 Master Mix 
25 µl
Universal PCR Primer/i5 Primer*, **
5 µl
Index (X) Primer /i7 Primer*, *** 
5 µl
Total Volume
50 µl


2.12.1B    Forward and Reverse Primers Combined

COMPONENT VOLUME PER ONE LIBRARY
Adaptor Ligated DNA (Step 2.11.9)   15 µl
(blue) NEBNext Ultra II Q5 Master Mix 
25 µl
Index (X)/Universal Primer Mix**** 
10 µl
Total Volume
50 µl


*The primers are provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600) Oligos for Illumina. For use with Dual Index Primers (NEB #E7600), look at the NEB #E7600 manual for valid barcode combinations and tips for setting up PCR reactions.
**For use with Dual Index Primers (NEB #E7600) use only one i5 Primer per reaction.
***For use with NEBNext Multiplex Oligos (NEB #E7335, #E7710, #E7730 or #E7500) use only one Index Primer per PCR reaction. For use with Dual Index Primers (NEB #E7600) use only one i7 Primer per reaction.
****The primers are provided in NEBNext Multiplex Oligos for Illumina (NEB #E6609). Please refer to the NEB #E6609 manual for valid barcode combinations and tips for setting up PCR reactions.

2.12.2.    Mix well by gently pipetting up and down 10 times. Quickly spin the tube in a microcentrifuge.

2.12.3.    Place the tube on a thermocycler with the heated lid set to 105°C and perform PCR amplification using the following PCR cycling conditions (refer to Table 2.12.3A and Table 2.12.3B):

Table 2.12.3A:

CYCLE STEP
TEMP TIME
CYCLES
Initial Denaturation  98°C
30 seconds 1
Denaturation
Annealing/Extension
98°C
65°C
10 seconds
75 Seconds
6–15*, **
Final Extension  65°C
5 minutes 1
Hold 4°C



*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.
 
Table 2.12.3B: Recommended PCR cycles based on total RNA input amount:

TOTAL RNA INPUT RECOMMENDED PCR CYCLES
1,000 ng 6–7
100 ng
10–11
10 ng 13–14
5 ng
14–15


Note: PCR cycles are recommended based on high quality Universal Human Reference Total RNA. It may require optimization based on the sample quality to prevent PCR over-amplification.

2.13.    Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads

2.13.1.    Vortex SPRIselect Beads or NEBNext Sample Purification Beads to resuspend.

2.13.2.    Add 45 μl (0.9X) of resuspended beads to the PCR reaction (~ 50 μl). Mix well on a vortex mixer or by pipetting up and down at least 10 times.

2.13.3.    Incubate for 5 minutes at room temperature.

2.13.4.    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 (about 5 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

2.13.5.    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.

2.13.6.    Repeat Step 2.13.5 once for a total of 2 washing steps.

2.13.7.    Air dry the beads for 5 minutes while the tube is on the magnetic rack with the lid open.

Caution: Do not overdry the beads. This may result in lower recovery of DNA.

2.13.8.    Remove the tube from the magnetic rack. Elute the DNA target from the beads by adding 23 μl 0.1X TE (provided) to the beads. Mix well on a vortex mixer or by pipetting up and down several times. Quickly spin the tube in a microcentrifuge and incubate for 2 minutes at room temperature. Place the tube in the magnetic rack until the solution is clear.

2.13.9.    Transfer 20 μl of the supernatant to a clean PCR tube, and store at –20°C.

2.14.    Assess Library Quality on a Bioanalyzer (Agilent DNA 1000 Chip)

2.14.1.    Run 1 μl library on a DNA 1000 chip. If the library yield is too low to quantify on this chip, please run the samples on a DNA High Sensitivity chip.

2.14.2.    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 2.13.9) to 50 μl with 0.1X TE buffer and repeat the SPRIselect Bead Cleanup Step (Section 2.13).

Figure 2.14.1: Example of RNA library size distribution on a Bioanalyzer.
Figure 2-14-1