Protocol for use with NEBNext mRNA Library Prep Reagent Set for Illumina (E6100)

Symbols
 
 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: Bioanalyzer traces of clean mRNA Fragments. The mRNA fragments should have a normal distribution with a peak at 200 nucleotides.
Figure 1.1: Bioanalyzer traces of clean mRNA Fragments. The mRNA fragments should have a normal distribution with a peak at 200 nucleotides.

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  (pink) ProtoScript II Reverse Transcriptase to the reaction.
  7. Incubate sample with the heated lid set to 105°C as follows:
    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 by gently pipetting up and down at least 10 times.
  3. Incubate for 5 minutes at room temperature.
  4. Place the tube/plate 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/plate 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-HCI. Mix well on a vortex mixer or by gently pipetting up and down several times, 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 1.5 ml microcentrifuge tube:
    Purified double-stranded cDNA 50 μl
    Nuclease-free Water 25 μl
     (green) 10X Phosphorylation Reaction Buffer 10 μl
    (green) Deoxynucleotide Solution Mix 4 μl
    (green) T4 DNA Polymerase 5 μl
    (green) E. coli DNA Polymerase I, Large (Klenow) Fragment 1 μl
    (green) T4 Polynucleotide Kinase 5 μl
    -----------------------------------------------
    Total volume 100 μl
  2. Incubate in a thermal cycler 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 by gently 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 sample 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/plate 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 from the beads into 40 μl 0.1X TE or 10 mM Tris-HCI. Mix well on a vortex mixer 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 (32 μl) and transfer to a clean 1.5 ml LoBind® (Eppendorf AG) 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 32 μl
     (yellow) NEBuffer2 5 μl
    (yellow) Deoxyadenosine 5´-Triphosphate (1 mM) 10 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.

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 μl). Mix well by gently 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 30 μl 0.1X TE or 10 mM Tris-HCI. Mix well by pipetting up and down several times, and incubate for 2 minutes at room temperature. 
  9. Put the tube in the magnetic stand until the solution is clear. Remove supernatant (23 μl) and transfer to a clean 1.5 ml LoBind® (Eppendorf AG) 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 23 μl
    (red) 2X Quick Ligation Reaction Buffer 25 μl
    (red) NEBNext Adaptors (15 μM)* 1 μl
    (red) Quick T4 DNA Ligase 1 μl
    -----------------------------------------------
    Total volume 50 μl
  2. * The NEBNext adaptor is provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E6609, #E7600) Oligos for Illumina. 

  3. Incubate 15 minutes at room temperature.
  4. Add 3 μl of  (red) 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, #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 Agencourt AMPure XP Beads to resuspend.
  2. Add 1.8X (90 μl) of resuspended Agencourt AMPure XP Beads to the ligation reaction (~50 μl). Mix well by gently pipetting up and down at least 10 times.
  3. Incubate for 5 minutes at room temperature.
  4. Place the tube/plate 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 from the beads into 155 μl of 0.1X TE or 10 mM Tris-HCI to the beads for bead-based size selection. Note: For size selection suing E-Gel size select gels, elute the DNA into 20 μl of 0.1X TE or 10 mM Tris-HCI.
  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.

1.13A 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. 

Note: X refers to the original sample volume of 150 μl.
  1. Add 135 μl (0.9X) resuspended Agencourt AMPure XP Beads to 150 μl eluted DNA from Step 9. Mix well by gently pipetting up and down at least 10 times.
  2. Incubate for 5 minutes at room temperature.
  3. Place the tube/plate 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 Agencourt 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 sample 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 from the magnet. Elute DNA target from beads into 22 μl of 10 mM Tris-HCI 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 an 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

Note: NEBNext Singleplex and Multiplex Oligos for Illumina (NEB #E7350, #E7335 and #E7500) now have new primer concentrations (10 μM). Please cleck oligo kit lot numbers to determine how to set up your PCR reaction. 

>Follow Section 1.14A if you using the following oligos (10 μM primer):
NEBNext Singleplex Oligos for Illumina (NEB #E7350) lot 0071412
NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335) lot 0091412
NEBNext Multiplex Oligos for Illumina (Set 2, NEB #E7500) lot 0071412
NEBNext Multiplex Oligos for Illumina (Dual Index Primers, NEB #E7600) all lots

Follow Section 1.14B if you are using NEBNext Multiplex Oligos for Illumina (96 Index Primers, NEB #E6609)

Follow Section 1.14C if you are using the following oligos (25 μM primer):
NEBNext Singleplex Oligos for Illumina (NEB #E7350) lots 0051402 or 0061410
NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335) lots 0071402 or 0081407
NEBNext Multiplex Oligos for Illumina (Set 2, NEB #E7500) lots 0051402 or 0061407

1.14A PCR Enrich Adaptor Ligated cDNA Library
  1. Mix the following components in sterile strip tubes:
  2. 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, #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 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. 
    *** For use with Dual Index Primers (NEB #E7600) use only one i5 Primer per reaction.
  3. PCR reaction conditions:
  4.  CYCLE STEP TEMP  TIME  CYCLES 
     Initial Denaturation 98°C  10 seconds 
     Denaturation
    Annealing/ Extension
    98°C
    65°C 
    10 seconds
    75 seconds 
    10-12* 
     Final Extension 65°C  5 minutes 
     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. 
  5. 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:
  2. 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. 
  3. PCR cycling conditions:
  4. CYCLE STEP  TEMP  TIME  CYCLES 
     Initial Denaturation 98°C  10 seconds 
     Denaturation
    Annealing/ Extension
    98°C
    65°C 
    10 seconds
    75 seconds 
    10-12* 
     Final Extension 65°C  5 minutes 
     Hold 4°C    

  5. Proceed to Purify using AMPure XP Beads in Section 1.15.
1.14C PCR Enrish Adaptor Ligated cDNA Library
  1. Mix the following components in sterile strip tubes:
  2. Size Selected cDNA    20 μl
     (blue) Index Primer*,**    1 μl
     (blue) Universal PCR Primer*    1 μl
     (blue) NEBNext Q5 Hot Start HiFi PCR Master Mix    25 μl
    Sterile H2O    3 μl
    ---------------------------------------------------------------------------------
    Total volume    50 μl
    * The primers are provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500) Oligos for Illumina.
    **For use with NEBNext Multiplex Oligos (NEB #E7335 or #E7500) use only one Index Primer per PCR reaction.
  3. PCR cycling conditions:
  4. CYCLE STEP  TEMP  TIME  CYCLES 
     Initial Denaturation 98°C  10 seconds 
     Denaturation
    Annealing/ Extension
    98°C
    65°C 
    10 seconds
    75 seconds 
    10-12* 
    Final Extension 65°C  5 minutes 
    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. 
  5. 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 gently 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 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.
  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.
  6. Repeat Step 5 once.
  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 target.
  8. Remove the plate from the magnet. Elute the DNA target from the beads into 23 μl 0.1X TE Buffer. Mix well by gently 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. 
      Figure 1.2: Example of mRNA Library size distribution on a Bioanalyzer.