Protocol for use with rRNA Depleted FFPE RNA and NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina® (E7760, E7765)

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

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

RNA Integrity:
RNA Integrity Number (RIN) is computed using ribosomal RNA (rRNA) amount in the sample. If rRNA is removed by any method, the RIN value should not be used to evaluate the integrity of the RNA sample. The following recommendation apply to the total RNA samples only.

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 4.

For highly degraded samples (RIN = 1 to 2) (e.g. FFPE), which does not require fragmentation, follow the library preparation protocol in Chapter 5 (current chapter).

RNA Purity:
The RNA sample should be free of DNA, salts (e.g., Mg2+, or guanidinium salts), divalent cation chelating agents (e.g. EDTA, EGTA, citrate), or organics (e.g., phenol and ethanol).

Starting Material: 1 ng – 100 ng alternative rRNA depleted FFPE RNA that is quantified after rRNA depletion. RNA should be DNA free in up to 5 μl of Nuclease-free Water, quantified by Qubit Fluorometer and quality checked by Bioanalyzer.

5.1.    Priming of Highly Degraded RNA (FFPE) Which has a RIN ≤ 2 and Does not Require Fragmentation

5.1.1.    Assemble the Priming Reaction on ice by adding the following components:

PRIMING REACTION VOLUME
rRNA Depleted FFPE RNA (1 ng–100 ng) 5 µl
(lilac) Random Primers
1 µl
Total Volume
6 µl

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

5.1.3.    Briefly spin down the samples in a microcentrifuge.

5.1.4.    Incubate the sample in a preheated thermal cycler as follows.
        5 minutes at 65°C, with heated lid set at 105°C
        Hold at 4°C


5.1.5.    Transfer the tube directly to ice and proceed to First Strand cDNA Synthesis.

5.2.    First Strand cDNA Synthesis

5.2.1.    Assemble the first strand synthesis reaction on ice by adding the following components:

FIRST STRAND SYNTHESIS REACTION VOLUME
Primed RNA (Step 5.1.5) 6 µl
(lilac) NEBNext First Strand Synthesis Reaction Buffer
4 µl
(brown) NEBNext Strand Specificity Reagent  8 µl
(lilac) NEBNext First Strand Synthesis Enzyme Mix  2 µl
Total Volume
20 µl


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

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

                         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

5.2.4.    Proceed directly to Second Strand cDNA Synthesis.

5.3.    Second Strand cDNA Synthesis

5.3.1    Assemble the second strand cDNA synthesis reaction on ice by adding the following components to the first strand reaction product from Step 5.2.4:

SECOND STRAND SYNTHESIS REACTION VOLUME
First Strand Synthesis Product (Step 5.2.4)
20 µl
(orange) NEBNext Second Strand Synthesis Reaction Buffer with dUTP (10X) 8µl
(orange) NEBNext Second Strand Synthesis Enzyme Mix  4 µl
Nuclease-free Water  48 µl
Total Volume
80 µl


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

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

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

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

5.4.2.    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.

5.4.3.    Incubate for 5 minutes at room temperature.

5.4.4.    Briefly spin the tube in a microcentrifuge to collect any sample on 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.

5.4.5.    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.

5.4.6.    Repeat Step 5.4.5 once for a total of 2 washing steps.  

5.4.7.    Air dry the beads for 5 minutes while the tube is on the magnet with lid open.

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

5.4.8.    Remove the tube from the magnet. 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.

5.4.9.    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.

5.5.    End Prep of cDNA Library

5.5.1.    Assemble the end prep reaction on ice by adding the following components to the second strand synthesis product from Step 5.4.9.

END PREP REACTION VOLUME
Second Strand Synthesis Product (Step 5.4.9)
50 µl
(green) NEBNext Ultra II End Prep Reaction Buffer  7µl
(green) NEBNext Ultra II End Prep Enzyme Mix  3 µl
Total Volume
60 µl


5.5.2.    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.

5.5.3.    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.

5.5.4.    Proceed immediately to Adaptor Ligation.

5.6.    Adaptor Ligation

5.6.1.      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.

PURIFIED RNA DILUTION REQUIRED
100 ng–11 ng
5-fold dilution in Adaptor Dilution Buffer
10 ng–1 ng  25-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.

5.6.2.    Assemble the ligation reaction on ice by adding the following components, in the order given, to the end prep reaction product from Step 5.5.4.

LIGATION REACTION VOLUME
End Prepped DNA (Step 5.5.4) 
60 µl
Diluted Adaptor (Step 5.6.1)  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.

5.6.3.    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.

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

5.6.5.    Add 3 μl (blue) USER Enzyme to the ligation mixture from Step 5.6.4, resulting in total volume of 96.5 μl.

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

5.6.7.    Proceed immediately to Purification of Ligation Reaction.

5.7.    Purification of Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads

5.7.1.    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.

5.7.2.    Incubate for 10 minutes at room temperature.

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

5.7.4.    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.

5.7.5.    Repeat Step 5.7.4 once for a total of 2 washing steps.  

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

5.7.7.    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.

5.7.8.    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 several times. Quickly spin the tube and incubate for 2 minutes at room temperature. Put the tube on the magnet until the solution is clear.

5.7.9.    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.


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

   Follow Section 5.8.1A 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 5.8.1B if you are using NEBNext Multiplex Oligos for Illumina (96 Index Primers, NEB #E6609).

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

5.8.1A    Forward and Reverse Primers Separate

COMPONENT VOLUME PER ONE LIBRARY
Adaptor ligated DNA (Step 5.7.9)   
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


5.8.1B    Forward and Reverse Primers Combined

COMPONENT VOLUME PER ONE LIBRARY
Adaptor ligated DNA (Step 5.7.9)
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.

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

5.8.3.    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 5.8.3A and Table 5.8.3B):

Table 5.8.3A:

CYCLE STEP
TEMP TIME
CYCLES
Initial Denaturation  98°C
30 seconds 1
Denaturation
Annealing/Extension
98°C
65°C
10 seconds
75 Seconds
7–13*, **
Final Extension  65°C
5 minutes 1
Hold 4°C



*The number of PCR cycles should be adjusted based on RNA input. The recommendation of PCR cycles are based on internal tests for FFPE RNA.
**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 5.8.3B: Recommended PCR cycles based on input amount:

rRNA DEPLETED FFPE RNA
 (QUANTIFIED AFTER rRNA DEPLETION) 
RECOMMENDED PCR CYCLES
100 ng
7-8
50 ng 8-9
10 ng 10-11
1 ng
13-14


Note: PCR cycles are recommended based on internally tested FFPE RNA. It may require optimization based on the sample quality to prevent PCR over-amplification.

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

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

5.9.2.    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.

5.9.3.    Incubate for 5 minutes at room temperature.

5.9.4.    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.

5.9.5.    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.

5.9.6.    Repeat Step 5.9.5 once for a total of 2 washing steps.

5.9.7.    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.

5.9.8.    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 on the magnetic rack until the solution is clear.

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

5.10.    Assess Library Quality on a Bioanalyzer (Agilent High Sensitivity Chip)

5.10.1.    Run 1 μl library on a DNA High Sensitivity Chip.

5.10.2.    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 5.9.9) to 50 μl with 0.1X TE Buffer and repeat the SPRIselect Bead or NEBNext Sample Purification Bead Cleanup Step (Section 5.9)

Figure 5.10.1: Example of FFPE RNA library size distribution on a Bioanalyzer.
Figure 5-10-1