Protocol for use with NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490) 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. For PolyA mRNA enrichment, high quality RNA with a RIN score > 7 is required.

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: 10 ng–1 µg total RNA quantified by Qubit® Fluorometer and quality checked by Bioanalyzer.
The protocol is optimized for approximately 200 bp RNA inserts. To generate libraries with longer RNA insert sizes, refer to Appendix A (Chapter 6) for recommended fragmentation times and size selection conditions.

Keep all the buffers on ice, unless otherwise indicated.

1.1. Preparation of First Strand Reaction Buffer and Random Primer Mix
 
1.1.1. Prepare the First Strand Synthesis Reaction Buffer and Random Primer Mix (2X) in a nuclease-free microcentrifuge tube as follows:

COMPONENT VOLUME
(lilac) NEBNext First Strand Synthesis Reaction Buffer
8 µl
(lilac) NEBNext Random Primers
2 µl
Nuclease-free water  
10 µl
Total Volume
20 µl


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

Note: Keep the mix on ice until mRNA is purified. It will be used in Step 1.2.36 and 1.2.39.

1.2. mRNA Isolation, Fragmentation and Priming Starting with Total RNA

1.2.1. Dilute the total RNA with nuclease-free water to a final volume of 50 μl in a nuclease-free 0.2 ml PCR tube and keep on ice.

1.2.2. To wash the Oligo dT Beads, add the following to a 1.5 ml nuclease-free tube. If preparing multiple libraries, beads for up to 10 samples can be added to a single 1.5 ml tube for subsequent washes.

COMPONENT VOLUME
Oligo dT Beads d(T)25
20 µl
RNA Binding Buffer (2X)
100 µl
Total Volume
120 µl


1.2.3. Wash the beads by pipetting up and down several times. 

1.2.4. Place the tube on the magnet and incubate at room temperature until the solution is clear (~2 minutes).

1.2.5. Remove and discard all of the supernatant from the tube. Take care not to disturb the beads. 

1.2.6. Remove the tube from the magnetic rack. 

1.2.7. Add 100 μl RNA Binding Buffer (2X) to the beads and wash by pipetting up and down. If preparing multiple libraries, add 100 µl RNA Binding Buffer (2X) per sample.

1.2.8. Place the tubes on the magnet and incubate at room temperature until the solution is clear (~2 minutes). 

1.2.9. Remove and discard the supernatant from the tube. Take care not to disturb the beads.

1.2.10. Add 50 μl RNA Binding Buffer (2X) to the beads and mix by pipetting up and down until beads are homogenous. If preparing multiple libraries, add 50 μl RNA Binding Buffer (2X) per sample.

1.2.11. Add 50 μl beads to each RNA sample from Step 1.2.1. Mix thoroughly by pipetting up and down several times.

1.2.12. Place the tube in a thermal cycler and close the lid. Heat the sample at 65°C for 5 minutes and cool to 4°C with the heated lid set at ≥ 75°C to denature the RNA and facilitate binding of the mRNA to the beads.

1.2.13. Remove the tube from the thermal cycler when the temperature reaches 4°C.

1.2.14. Mix thoroughly by pipetting up and down several times. Place the tube on the bench and incubate at room temperature for 5 minutes to allow the mRNA to bind to the beads.

1.2.15. Place the tube on the magnetic rack at room temperature until the solution is clear (~2 minutes). 

1.2.16. Remove and discard all of the supernatant. Take care not to disturb the beads. 

1.2.17. Remove the tube from the magnetic rack.

1.2.18. Wash the beads by adding 200 μl of Wash Buffer to the tube to remove unbound RNA. Gently pipette the entire volume up and down 6 times to mix thoroughly. 

1.2.19. Place the tube on the magnetic rack at room temperature until the solution is clear (~2 minutes).

1.2.20. Remove and discard all of the supernatant from the tube. Take care not to disturb the beads. 

1.2.21. Remove the tube from the magnetic rack. 

1.2.22. Repeat steps 1.2.18–1.2.21.

1.2.23. Add 50 μl of Tris Buffer (provided) to each tube. Gently pipette up and down 6 times to mix thoroughly.

1.2.24. Place the tube on the thermal cycler. Close the lid and heat the samples at 80°C for 2 minutes, then hold at 25°C with the heated lid set at ≥ 90°C to do the first elution of the mRNA from the beads. 

1.2.25. Remove the tube from the thermal cycler when the temperature reaches 25°C

1.2.26. Add 50 μl of RNA Binding Buffer (2X) to the sample to allow the mRNA to re-bind to the beads. Mix thoroughly by gently pipetting up and down several times.

1.2.27. Incubate the tube at room temperature for 5 minutes.

1.2.28. Place the tube on the magnetic rack at room temperature until the solution is clear (~2 minutes).

1.2.29. Remove and discard the supernatant from the tube. Take care not to disturb the beads.

1.2.30. Remove the tube from the magnetic rack.

1.2.31. Wash the beads by adding 200 μl of Wash Buffer. Gently pipette the entire volume up and down 6 times to mix thoroughly. 

1.2.32. Spin down the tube briefly to collect the liquid from the wall and lid of the tube.

Note: It is important to spin down the tube to prevent carryover of the Wash Buffer in subsequent steps.
 

1.2.33 Place the tube on the magnet at room temperature until the solution is clear (~2 minutes).

1.2.34. Remove and discard all of the supernatant from the tube. Take care not to disturb the beads that contains the mRNA.

Note: It is important to remove all of the supernatant to successfully fragment the mRNA in the subsequent steps.
 

1.2.35. Remove the tube from the magnetic rack.
 
  The next step provides a fragmentation incubation time resulting in an RNA insert size of ~ 200 nt. For RNA insert sizes > 200 nt, refer to Chapter 6 (Appendix A) for recommended fragmentation times in Step 1.2.37.

1.2.36. To elute the mRNA from the beads and fragment, add 11.5 μl of the First Strand Synthesis Reaction Buffer and Random Primer Mix (2X) prepared in Step 1.1.2, pipette up and down several times to resuspend the beads. 

1.2.37 Incubate the sample in a thermal cycler with the heated lid set at 105°C as follows:
15 minutes at 94°C
Hold at 4°C*
*Immediately transfer the tube to ice for 1 minute as soon as it is cool enough to handle (~65°C)

1.2.38. Quickly spin down the tube in a microcentrifuge to collect the liquid from the sides of the tube and place on the magnet right away until the solution is clear (~1-2 minutes). 

1.2.39. Collect the fragmented mRNA by transferring 10 μl of the supernatant to a nuclease-free 0.2 ml PCR tube.

Note 1: If the supernatant volume recovered is less than 10 μl for any reason, bring the volume up to 10 μl by adding the First Strand Synthesis Reaction Buffer and Random Primer Mix (2X) prepared in Step 1.1.2 and continue with the protocol.

Note 2: Avoid transferring the magnetic beads.


1.2.40. Place the tube on ice and proceed directly to First Strand cDNA Synthesis.   

1.3 First Strand cDNA Synthesis

1.3.1. Assemble the first strand cDNA synthesis reaction on ice by adding the following components into fragmented and primed RNA from Step 1.2.40.

FIRST STRAND cDNA SYNTHESIS REACTION VOLUME
Fragmented and primed RNA (Step 1.2.40) 10 µl
Nuclease-free Water 8 µl
(lilac) NEBNext First Strand Synthesis Enzyme Mix 2 µl
Total Volume
20 µl


1.3.2. Mix thoroughly by pipetting up and down several times.
1.3.3. 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

1.3.4. Immediately, perform Second Strand cDNA Synthesis.

1.4 Second Strand cDNA Synthesis


1.4.1. Assemble the second strand cDNA synthesis reaction on ice by adding the following components into the first strand synthesis reaction product from Step 1.3.4.

SECOND STRAND SYNTHESIS REACTION VOLUME
First-Strand Synthesis Product (Step 1.3.4) 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


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

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

1.5 Purification of Double-stranded cDNA using SPRIselect Beads or NEBNext Sample Purification Beads


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

1.5.2. 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.

1.5.3. Incubate for 5 minutes at room temperature.

1.5.4. Briefly spin the tube in a microcentrifuge to collect any sample from the sides of the tube. 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 DNA.

1.5.5. Add 200 μl of freshly prepared 80% ethanol to the tube while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.

1.5.6. Repeat Step 1.5.5 once for a total of 2 washing steps.

1.5.7. 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.

1.5.8. Remove the tube from the magnet. Elute the DNA target 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. Briefly spin the tube and incubate for 2 minutes at room temperature. Place the tube on the magnetic rack until the solution is clear. 

1.5.9. 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.

1.6 End Prep of cDNA Library

1.6.1. Assemble the end prep reaction on ice by adding the following components to second strand synthesis product from Step 1.5.9.

END PREP REACTION VOLUME
Second Strand cDNA Synthesis Product (Step 1.5.9)
50 µl
(green) NEBNext Ultra II End Prep Reaction Buffer
7 µl
(green) NEBNext Ultra II End Prep Enzyme Mix 3 µl
Total Volume
60 µl


1.6.2       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.

1.6.3. 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

1.6.4. Proceed immediately to Adaptor Ligation.

1.7 Adaptor Ligation


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

TOTAL RNA input   DILUTION REQUIRED
1,000 ng–250 ng 5-fold dilution in Adaptor Dilution Buffer
249 ng–100 ng
25-fold dilution in Adaptor Dilution Buffer
99 ng–10 ng  100-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.

1.7.2. Assemble the ligation reaction on ice by adding the following components, in the order given, to the end prep reaction product from Step 1.6.4.



LIGATION REACTION
VOLUME PER ONE LIBRARY
End Prepped DNA (Step 1.6.4) 60 µl
Diluted Adaptor (Step 1.7.1)    
2.5 µl
(red) NEBNext Ligation Enhancer  1 µl
(red) NEBNext Ultra II Ligation Master Mix   30 µl
Total Volume
93.5 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.

1.7.3. 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.

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

1.7.5 Add 3 μl (red) USER Enzyme to the ligation mixture from Step 1.7.4, resulting in total volume of 96.5 μl.

  Note: Steps 1.7.5 and 1.7.6 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.

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

1.7.7 Proceed immediately to Purification of the Ligation Reaction.

1.8 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.

1.8.1. 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.

1.8.2. Incubate for 5 minutes at room temperature.

1.8.3. 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 contain unwanted fragments (Caution: do not discard the beads).

1.8.4. 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.

1.8.5. Repeat Step 1.8.4 once for a total of 2 washing steps.

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

1.8.7. 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 target.

1.8.8. Remove the tube from the magnet. 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 magnetic rack until the solution is clear.

1.8.9. 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.


1.9. PCR Enrichment of Adaptor Ligated DNA

Follow Section 1.9.1A if 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 1.9.1B if you are using NEBNext Multiplex Oligos for Illumina (96 Index Primers, NEB #E6609).

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

1.9.1A. Forward and Reverse Primers Separate

COMPONENT
VOLUME PER ONE LIBRARY
Adaptor Ligated DNA (Step 1.8.9)  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


1.9.1B. Forward and Reverse Primers Combined

COMPONENT
VOLUME PER ONE LIBRARY
Adaptor Ligated DNA (Step 1.8.9)   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.

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

1.9.3. 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 1.9.3A and Table 1.9.3B):

Table 1.9.3A: 

CYCLE STEP
TEMP TIME
CYCLES
Initial Denaturation  98°C
30 seconds 1
Denaturation
Annealing/Extension
98°C

65°C
10 seconds

75 Seconds

7–15*, **
Final Extension  65°C
5 minutes 1
Hold 4°C



* The number of PCR cycles should be adjusted based on RNA input (Table 1.9.3B). 
** 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 1.9.3B:

TOTAL RNA INPUT
RECOMMENDED PCR CYCLES
1,000 ng 7–8
100 ng
11–12
10 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.

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


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

1.10.2. 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.

1.10.3. Incubate for 5 minutes at room temperature.

1.10.4. 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.

1.10.5. 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.

1.10.6. Repeat Step 1.10.5 once for a total of 2 washing steps.

1.10.7. 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.

1.10.8. 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.

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

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


1.11.1. 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. 

1.11.3. 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 1.10.9) to 50 μl with 0.1X TE buffer and repeat the SPRIselect Bead or NEBNext Sample Purification Bead Cleanup Step (Section 1.10).
Figure 1.11.1: Example of RNA library size distribution on a Bioanalyzer.

Fig 1 -11