Protocol for use with NEBNext mRNA Library Prep Master Mix Set for Illumina (E6110)

 This caution sign signifies a step in the protocol that has multiple paths leading to the same end point but is dependent on a user variable, like the amount of input DNA.

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

Starting Material: Purified mRNA (50–250 ng)

1.1 mRNA Fragmentation Protocol
  1. Mix the following components in a sterile PCR tube:
    Purified mRNA 1–18 μl
     (white) 10X RNA Fragmentation Reaction Buffer 2 μl
    Nuclease-free Water variable
    -----------------------------------------------
    Total Volume 20 μl
  2. Incubate in a preheated thermal cycler for 5 minutes at 94°C. This is the optimal condition for eukaryotic mRNA to generate 200 nucleotide RNA fragments (see Figure 1.1). Other types of mRNA may require optimizing incubation time to obtain desired fragment size distribution.
  3. Transfer tube to ice.
  4. Add 2 μl (white) 10X RNA Fragmentation Stop Solution.
1.2 RNA Purification after RNA Fragmentation using Agencourt RNAClean XP
  1. Add 28 μl of the nuclease-free water to the 22 μl fragmented RNA from Step 4 of the mRNA Fragmentation Protocol.
  2. Add 2.2X (110 μl) Agencourt RNAClean XP Beads and mix by pipetting up and down.
  3. Incubate samples on ice for 15 minutes.
  4. Place the tube on an appropriate magnetic rack to separate beads from the supernatant.
  5. When the solution is clear (about 5 minutes), discard the supernatant.
  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
  7. Repeat Step 6 once for a total of 2 washes.
  8. Briefly spin the tube, and put the tube back in the magnetic rack.
  9. Completely remove the residual ethanol, and air dry the beads.
  10. Remove the tube from the magnetic rack. Elute RNA from the beads with 15 μl nuclease-free water.
  11. Mix well by pipetting up and down, and put the tube in the magnetic rack until the solution is clear.
  12. Use 1 μl of the supernatant to QC the fragment size distribution on an Agilent Bioanalyzer RNA pico chip (Section 1.3).
  13. Transfer 13.5 μl of the supernatant to a clean PCR tube.
  14. Place the sample on ice and proceed with first strand cDNA synthesis in Section 1.4.
1.3 Assess the Yield and the Size Distribution of the Fragmented mRNA

Run 1 μl in the Agilent Bioanalyzer® 2100 (Agilent Technologies, Inc.) using a RNA Pico chip.

Note: Clean mRNA fragments show a distinct band on the Bioanalyzer (Figure 1.1).

Figure 1.1: Relative size distribution of eukaryotic mRNA fragments as seen using the Bioanalyzer 2100.


1.4 First Strand cDNA Synthesis
  1. Mix the following components in a sterile PCR tube:
    Fragmented mRNA 13.5 μl
     (pink) Random Primers 1 μl
    -----------------------------------------------
    Total volume 14.5 μl
  2. Incubate in a preheated thermal cycler for 5 minutes at 65°C.
  3. Spin tube briefly and place on ice
  4. To the fragmented mRNA and Random Primers add:
     (pink) 5X First Strand Synthesis Reaction Buffer 4 μl
    (pink) Murine RNase Inhibitor 0.5 μl
    -----------------------------------------------
    Total volume 19 μl
  5. Incubate in a preheated thermal cycler for 2 minutes at 25°C.
  6. Add 1 μl ProtoScript II Reverse Transcriptase to the reaction.
  7. Incubate sample as follows with the heated lid set to 105°C:
    10 minutes at 25°C
    50 minutes at 42°C
    15 minutes at 70°C
    Hold at 4°C
  8. Place the tube on ice.
1.5 Second Strand cDNA Synthesis
  1. Add the following reagents to the First Strand Synthesis reaction:
    Nuclease-free Water 48 μl
     (orange) 10X Second Strand Synthesis Reaction Buffer 8 μl
    (orange) Second Strand Synthesis Enzyme Mix 4 μl
    -----------------------------------------------
    Total volume 80 μl
  2. Mix thoroughly by gentle pipetting.
  3. Incubate in a thermal cycler for 2.5 hours at 16°C.
    Note: If you need to stop at this point in the protocol after the 2.5 hours incubation at 16°C, samples can be left in the thermal cycler overnight at 4°C.
1.6 Purify the double-stranded cDNA using 1.8X Agencourt AMPure XP Beads
  1. Vortex AMPure XP Beads to resuspend.
  2. Add 1.8X (144 μl) of resuspended AMPure XP 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.
  3. Incubate for 5 minutes at room temperature.
  4. Place the tube on an appropriate magnetic stand to separate beads from 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.
  5. Add 200 μl of freshly prepared 80% ethanol to the tube while in the magnetic stand. Mix by pipetting up and down. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
  6. Repeat Step 5 once.
  7. Air dry the beads for 5 minutes while the tube is on the magnetic stand with lid open.
    Caution: Do not overdry the beads. This may result in lower recovery of DNA target.
  8. Remove the tube from the magnet. Elute the DNA target from the beads into 55 μl 0.1X TE or 10 mM Tris-HCl. Mix well on a vortex mixer or by pipetting up and down, and incubate for 2 minutes at room temperature.
  9. Put the tube in the magnetic stand until the solution is clear. Remove supernatant (50 μl) and transfer to a clean 1.5 ml LoBind® (Eppendorf AG) tube.
1.7 End Repair of cDNA Library

  1. Mix the following components in a sterile 1.5 ml microcentrifuge tube:
    Purified double-stranded cDNA 50 μl
    Nuclease-free Water 35 μl
     (green) NEBNext End Repair Reaction Buffer 10 μl
     (green) NEBNext End Repair Enzyme Mix 5 μl
    -----------------------------------------------
    Total volume 100 μl
  2. Incubate in a heat block for 30 minutes at 20°C.
1.8 Purify the end-repaired cDNA using 1.8X Agencourt AMPure XP Beads. Note: X refers to original sample volume.

  1. Vortex AMPure XP Beads to resuspend.
  2. Add 1.8X (180 μl) of resuspended AMPure XP Beads to the end-repaired DNA (~100 μl). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
  3. Incubate for 5 minutes at room temperature.
  4. Place the tube on an appropriate magnetic stand to separate beads from 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.
  5. Add 200 μl of freshly prepared 80% ethanol to the tube while in the magnetic stand. Mix by pipetting up and down. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
  6. Repeat Step 5 once.
  7. Air dry the beads for 5 minutes while the tube is on the magnetic stand with lid open.
    Caution: Do not overdry the beads. This may result in lower recovery of DNA target.
  8. Remove the tube from the magnet. Elute the DNA target from the beads into 46 μl 0.1X TE or 10 mM Tris-HCl. Mix well on a vortex mixer or by pipetting up and down, and incubate for 2 minutes at room temperature.
  9. Put the tube in the magnetic stand until the solution is clear. Remove supernatant (42 μl) and transfer to a clean 1.5 ml LoBind tube.

1.9 dA-Tailing of cDNA Library

  1. Mix the following components in a sterile 1.5 ml microcentrifuge tube:
    Purified, End Repaired cDNA 42 μl
     (yellow) 10X NEBNext dA-Tailing Reaction Buffer 5 μl
     (yellow) Klenow Fragment (3´→5´ exo) 3 μl
    -----------------------------------------------
    Total volume 50 μl
  2. Incubate in a heat block for 30 minutes at 37°C with the heated lid set to 42°C.

1.10 Purify the dA-Tailed DNA using 1.8X Agencourt AMPure XP Beads

  1. Vortex AMPure XP Beads to resuspend.
  2. Add 1.8X (90 μl) of resuspended AMPure XP Beads to the dA tailed DNA (~50 μ l). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
  3. Incubate for 5 minutes at room temperature.
  4. Place the tube on an appropriate magnetic stand to separate beads from 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.
  5. Add 200 μl of freshly prepared 80% ethanol to the tube while in the magnetic stand. Mix by pipetting up and down. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
  6. Repeat Step 5 once.
  7. Air dry the beads for 5 minutes while the tube is on the magnetic stand with the lid open.
    Caution: Do not overdry the beads. This may result in lower recovery of DNA target.
  8. Remove the tube from the magnet. Elute the DNA target from the beads into 42 μl 0.1X TE or 10 mM Tris-HCl. Mix well on a vortex mixer or by pipetting up and down, and incubate for 2 minutes at room temperature.
  9. Put the tube in the magnetic stand until the solution is clear. Remove supernatant (38 μl) and transfer to a clean 1.5 ml LoBind tube.

1.11 Adaptor Ligation of cDNA Library

  1. Mix the following components in a sterile 1.5 ml microcentrifuge tube:
    Purified, dA-Tailed cDNA 38 μl
     (red) 5X NEBNext Quick Ligation Reaction Buffer 10 μl
     (red) NEBNext Adaptor (15 μM)* 1 μl
     (red) Quick T4 DNA Ligase 1 μl
    -----------------------------------------------
    Total volume 50 μl
    * The NEBNext adaptor is provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E6609, #E7600) Oligos for Illumina.
  2. Incubate 15 minutes at room temperature.
  3. Add 3 μl of USER™ enzyme, mix by pipetting up and down, and incubate at 37°C for 15 minutes.

    NOTE: This step is only for use with NEBNext adaptors. USER enzyme can be found in the NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E6609, #E7600) Oligos for Illumina.

    A precipitate can form upon thawing of the NEBNext Q5 Hot Start HiFi PCR Master Mix. To ensure optimal performance, place the master mix at room temperature while performing cleanup of adaptor-ligated DNA. Once thawed, gently mix by inverting the tube several times.

1.12 Cleanup of Adaptor-Ligated DNA

  1. Vortex AMPure XP Beads to resuspend.
  2. Add 90 μl of resuspended AMPure XP Beads to the ligation reaction (~53 μl). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
  3. Incubate for 5 minutes at room temperature.
  4. Place the tube on an appropriate magnetic stand to separate beads from 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.
  5. Add 200 μl of freshly prepared 80% ethanol to the tube while in the magnetic stand. Mix by pipetting up and down. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
  6. Repeat Step 5 once.
  7. Air dry the beads for 5 minutes while the tube is on the magnetic stand with lid open.
    Caution: Do not overdry the beads. This may result in lower recovery of DNA target.
  8. Remove the tube/plate from the magnet. Elute the DNA target by adding 155 μl of 10 mM Tris-HCl or 0.1 X TE to the beads for bead-based size selection.
    Note: For size selection using E-Gel size select gels, elute the DNA into 20 μl 0.1X TE or 10 mM Tris-HCl.
  9. Mix well on a vortex mixer or by pipetting up and down and incubate for 2 minutes at room temperature.
  10. Put the tube/PCR plate in the magnetic stand until the solution is clear. Remove supernatant (150 μl) and transfer to a clean 1.5 ml LoBind tube.

1.13 Size Selection of Adaptor-ligated DNA using Agencourt AMPure XP Beads

The following size selection protocol is for libraries with 200 bp inserts only. For libraries with larger fragment inserts, please optimize bead: DNA ratio accordingly.

  1. Add 135 μl (0.9X) resuspended AMPure XP Beads to 150 μl eluted DNA from step 9. Mix well on a vortex mixer or by pipetting up and down at least 10 times.
  2. Incubate for 5 minutes at room temperature.
  3. Place the tube on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully transfer the supernatant to a new tube (Caution: do not discard the supernatant). Discard beads that contain the large fragments.
  4. Add 30 μl (0.2X) resuspended AMPure XP Beads to the supernatant, mix well and incubate for 5 minutes at room temperature.
  5. Put the tube on an appropriate magnetic stand to separate beads from 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 (Caution: do not discard beads).
  6. 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.
  7. Repeat Step 6 once.
  8. Briefly spin the tube, and put the tube back in the magnetic stand.
  9. Completely remove the residual ethanol, and air dry beads for 5 minutes while the tube is on the magnetic stand with lid open.
    Caution: Do not overdry the beads. This may result in lower recovery of DNA target.
  10. Remove the tube/plate from the magnet. Elute the DNA target from the beads by adding 22 μl of 10 mM Tris-HCl or 0.1X TE.
  11. Mix well on a vortex mixer or by pipetting up and down and incubate for 2 minutes at room temperature.
  12. Put the tube/PCR plate in the magnetic stand until the solution is clear. Transfer 20 μl of the supernatant to a clean PCR tube, and proceed to PCR enrichment.
Adaptor ligated DNA can also be size selected using a E-Gel size select gel. After size selection, purify DNA sample on one QIAquick column and elute in 22 μl of nuclease free water.


1.14 PCR Enrichment of Adaptor Ligated DNA

Follow Section 1.14A if you are using the following oligos (10 μM primer):
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 1.14B if you are using NEBNext Multiplex Oligos for Illumina (96 Index Primers, NEB #E6609)

1.14A PCR Enrich Adaptor Ligated cDNA Library

  1. Mix the following components in sterile strip tubes:
    Size Selected cDNA 20 μl
    (blue) Index Primer/i7 Primer*,** 2.5 μl
    (blue) Universal PCR Primer/i5 Primer*,*** 2.5 μl
    (blue) NEBNext Q5 Hot Start HiFi PCR Master Mix 25 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 the PCR reaction.
    ** For use with NEBNext Multiplex Oligos (NEB #E7335, #E7500, #E7710, #E7730) use only one index primer per PCR reaction. For use with Dual Index Primers (NEB #E7600) use only one i7 Primer per reaction.
    *** For use with Dual Index Primers (NEB #E7600 ) use only one i5 Primer per reaction.
  2. PCR cycling conditions:
    CYCLE STEP TEMP TIME CYCLES
    Initial Denaturation 98°C 10 seconds 1
    Denaturation 98°C 10 seconds 10–12*
    Annealing/Extension 65°C 75 seconds
    Final Extension 65°C 5 minutes 1
    Hold 4°C  
    * The number of PCR cycles should be adjusted based on mRNA input. If 50 ng of purified mRNA is the starting input, it is recommended to perform 12 cycles of PCR.
  3. Proceed to Purify using Ampure XP Beads in Section 1.15
1.14B PCR Enrich Adaptor Ligated cDNA Library

  1. Mix the following components in sterile strip tubes:
    Size Selected cDNA 20 μl
    (blue) Index/Universal Primer Mix* 5 μl
    (blue) NEBNext Q5 Hot Start HiFi PCR Master Mix 25 μl
    -----------------------------------------------
    Total volume 50 μl
    * 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. PCR cycling conditions:
    CYCLE STEP TEMP TIME CYCLES
    Initial Denaturation 98°C 10 seconds 1
    Denaturation 98°C 10 seconds 10–12*
    Annealing/Extension 65°C 75 seconds
    Final Extension 65°C 5 minutes 1
    Hold 4°C  
    * The number of PCR cycles should be adjusted based on mRNA input. If 50 ng of purified mRNA is the starting input, it is recommended to perform 12 cycles of PCR.
  3. Proceed to Purify using Ampure XP Beads in Section 1.15
1.15 Purify using AMPure XP Beads
    1. Vortex AMPure XP Beads to resuspend.
    2. Add 60 μl (1.2X) of resuspended AMPure XP Beads to the PCR reaction (~ 50 μl). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
    3. Incubate for 5 minutes at room temperature.
    4. Place the tube on an appropriate magnetic stand to separate beads from 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.
    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.
    6. Repeat Step 5 once.
    7. Air dry the beads for 5 minutes while the tube is on the magnetic stand with the lid open. Caution: Do not overdry the beads. This may result in lower recover of DNA target.
    8. Remove the tube from the magnet. Elute the DNA target from the beads into 23 μl 0.1X TE Buffer. Mix well on a vortex mixer or by pipetting up and down, and incubate for 2 minutes at room temperature. Put the tube in the magnetic stand until the solution is clear.
    9. Transfer 20 μl of the supernatant to a clean 1.5 ml LoBind tube. Libraries can be stored at –20°C.
    1.16 Assess Library Quality on a Bioanalyzer (High Sensitivity Chip)
    Check that the electropherogram shows a narrow distribution with a peak size approximately 270 bp.

    Figure 1.2: Example of mRNA Library size distribution on a Bioanalyzer.