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  • Protocol (E6110) for Use with NEBNext Singleplex (#E7350) or Multiplex (#E7335, #E7500) Oligos for Illumina

    Protocol

    Starting Material: Purified mRNA (50–250 ng)


    mRNA Fragmentation Protocol
    1. Mix the following components in a sterile PCR tube:
      Purified mRNA 1-18 μl
      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. Other types of mRNA may require optimizing incubation time to obtain desired fragment size distribution.
    3. Transfer tube to ice.
    4. Add 2 μl 10X RNA Fragmentation Stop Solution.
    Clean Up Fragmented RNA Using RNeasy MinElute Spin Columns (Strongly Recommended)
    1. Add 78 μl of the nuclease-free water to the 22 μl fragmented RNA from step 4. Purify sample using RNeasy® MinElute® Cleanup Kit (Qiagen #74204) following manufacturer instructions. Elute in 15.5 μl nuclease-free water. The recovered volume should be ~14.5 μl.

      Note: column purification removes short RNA Fragments and enriches the sample for RNA fragments longer than 200 nucleotides.
    Alternatively, Clean Up Fragmented RNA Using Ethanol Precipitation
    1. Mix the following components in a sterile 1.5ml microcentrifuge tube:
      Fragmented RNA from Step 4 22 μl
      3M Sodium Acetate, pH 5.5 2 μl
      Linear Acrylamide, 10 mg/ml 1-2 μl
      100% Ethanol 60 μl
      Total volume 85-86
    2. Incubate at -80°C or in a dry ice/methanol bath for 1 hour
    3. Centrifuge at 14,000 rpm for 25 minutes at 4°C in a microcentrifuge.
    4. Carefully remove ethanol.
    5. Wash pellet with 300 μl of freshly prepared 70% ethanol. Carefully pipette up and down the pellet. Make sure the pellet does not get stuck in the tip.
    6. Centrifuge at 14,000 rpm for 5 minutes at 4°C in a microcentrifuge. Carefully remove all 70% ethanol.
    7. Repeat steps 5 and 6 twice.

      Note: Insufficient pellet washing results in inhibition of the first strand cDNA synthesis due to carryover of magnesium and EDTA. If the Bioanalyzer traces of the mRNA fragments show a noisy baseline repeat the ethanol precipitation step and pellet washing steps.

    8. Air dry pellet for up to 10 minutes at room temperature (or longer if necessary) to remove residual ethanol.
    9. Resuspend in 14.5 μl Nuclease-free Water.

    First Strand cDNA Synthesis
    1. Mix the following components in a sterile PCR tube:
      Fragmented mRNA 13.5 μl
      Random Primers 1 μl
      Total volume 14.5 μl
    2. Incubate in a preheated thermal cycler for 5 minutes at 65C.
    3. Spin tube briefly and place on ice.
    4. To the fragmented mRNA and Random Primers add:
      5X First Strand Synthesis Reaction Buffer 4 μl
      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:
      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.

    Second Strand cDNA Synthesis
    1. Add the following reagents to the First Strand Synthesis reaction:
      Nuclease-free Water 48 μl
      10X Second Strand Synthesis Reaction Buffer 8 μl
      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.
    Purify the double-stranded cDNA using 1.8X Agencourt AMPure® XP Beads (Beckman Coulter, Inc.)(Recommended).
    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. 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 10 minutes while the tube is on the magnetic stand with lid open.
    8. Elute the DNA target from the beads into 55 μl water. Mix well on a vortex mixer or by pipetting up and down, and 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 tube.

      Alternatively, purify the double-stranded cDNA using QIAquick PCR Column Purification Kit and elute in 52 μl nuclease-free water.

    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
      NEBNext End Repair Reaction Buffer 10 μl
      NEBNext End Repair Enzyme Mix 5 μl
      Total volume 100 μl
    2. Incubate in a heat block for 30 minutes at 20°C.
    Purify the end-repaired cDNA using 1.8X Agencourt AMPure XP Beads (Recommended).
    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 10 minutes while the tube is on the magnetic stand with lid open.
    8. Elute the DNA target from the beads into 46 μl water. Mix well on a vortex mixer or by pipetting up and down, and 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® (Eppendorf AG)tube.

      Alternatively, purify the double-stranded cDNA using QIAquick PCR Column Purification Kit and elute in 42 μl nuclease free water.

    dA-Tailing of cDNA Library
    1. Mix the following components in a sterile 1.5 ml microcentrifuge tube:
      Purified, End Repaired cDNA 42 μl
      10X NEBNext dA-Tailing Reaction Buffer 5 μl
      Klenow Fragment (3´→5´ exo) 3 μl
      Total volume 50 μl
    2. Incubate in a heat block for 30 minutes at 37°C.
    Purify the dA-Tailed DNA using 1.8X Agencourt AMPure XP Beads (Recommended).
    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 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 10 minutes while the tube is on the magnetic stand with the lid open.
    8. Elute the DNA target from the beads into 42 μl water. Mix well on a vortex mixer or by pipetting up and down, and 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.

      Alternatively, purify the double-stranded cDNA using QIAquick PCR Column Purification Kit and elute in 40 μl nuclease free water.

    Adaptor Ligation of cDNA Library
    1. Mix the following components in a sterile 1.5 ml microcentrifuge tube:
      Purified, dA-Tailed cDNA 38 μl
      5X NEBNext Quick Ligation Reaction Buffer 10 μl
      NEBNext Adaptor (15 μM) 1 μl
      Quick T4 DNA Ligase 1 μl
      Total volume 50 μl
    2. Incubate 15 minutes at room temperature.
    3. 3. Add 3 μl of USER™ enzyme, mix by pipetting up and down, and incubate at 37°C for 15 minutes.
    Purify using AMPure XP Beads (strongly recommended).
    1. Vortex AMPure XP beads to resuspend.
    2. Add 1.8X (90 μl) of resuspended AMPure XP beads to the ligation 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. 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 10 minutes while the tube is on the magnetic stand with lid open.
    8. Elute the DNA target from the beads into 155 μl water for bead-based size selection or into 20 μl water for size selection using E-Gel® (Life Technologies, Inc.) size select gels. Mix well on a vortex mixer or by pipetting up and down, and put the tube in the magnetic stand until the solution is clear.
    9. Remove supernatant (150 μl) and transfer to a clean 1.5 ml LoBind tube.
    Size Selection of Adaptor-ligated DNA using Agencourt AMPure XP Beads or Invitrogen E-Gels (Strongly Recommended)
      Caution: 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 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 10 minutes while the tube is on the magnetic stand with lid open.
    10. Elute DNA target from beads into 25 μl nuclease free water. Mix well on a vortex mixer or by pipetting up and down, and put the tube in the magnetic stand until the solution is clear.
    11. Transfer 23 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 25 μl of nuclease free water.
    Alternatively, Size Selection of Adaptor-ligated DNA can be Performed Using a 2% Agarose Gel
    1. Purify the adaptor-ligated DNA using QIAGEN MinElute PCR Purification Kit and elute in 12 μl of nuclease free water.
      IMPORTANT: Before eluting the DNA from the column, centrifuge the column with the lid of the spin column open for 5 minutes at 13,200 rpm. Centrifugation with the lid open ensures that no ethanol is carried over during DNA elution. Residual ethanol interferes with the correct loading of the sample on the gel.
    2. Pour a 5 mm thick 2% agarose gel with a 1mm thick gel space and allow to cool.
    3. Run adaptor ligated cDNA for 2 hours at 150 V or until the dye front has traveled 10 cm.
    4. Isolate the desired cDNA fragment from the gel.
    5. Purify DNA from the gel slice using a QIAquick Gel Extraction Purification Kit, purifying the sample on one column and elute in 25 μl of nuclease free water.

    PCR Enrich Adaptor Ligated cDNA Library
    1. Mix the following components in a sterile PCR tube:
      Size Selected cDNA 23 μl
      NEBNext High-Fidelity 2X PCR Master Mix** 25 μl
      Universal PCR Primer (25 M) 1 μl
      Index Primer (X)* (25 M) 1 μl
      Total volume 50 μl
      *If you are using the NEBNext Multiplex Oligos for Illumina (Index Primers 1-12), for each reaction only one of the 12 PCR primer Indices Is used during the PCR step.
      **NEBNext High-Fidelity 2X PCR Master Mix will be replacing Phusion High-Fidelity PCR Master Mix. Both vials will be supplied for a limited time only.
    2. PCR cycling conditions:
      Cycle Step Temperature Time Cycles
      Initial Denaturation 98°C 10 seconds 1
      Denaturation 98°C 10 seconds 10-12*
      Annealing 65°C 30 seconds
      Extension 72°C 30 seconds
      Final Extension 72°C 5 minutes 1
      4°C hold
      *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.
    Purify using AMPure XP Beads (strongly recommended)
    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.
    8. 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 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, and store at -20°C.

      Alternatively, purify the PCR enriched cDNA using a QIAquick PCR Column Purification Kit and elute in 20 μl of sterile water or elution buffer.
    Assess Library Quality on a Bioanalyzer (High Sensitivity Chip)

    Check that the electropherogram shows a narrow distribution with a peak size approximately 270 bp.