Protocol for use with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760, E7765)

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

A	B
Component	Volume
NEBNext First Strand Synthesis Reaction Buffer	8 µl
NEBNext Random Primers	2 µl
Nuclease-free Water	10 µl
Total Volume	20 µl

You can prepare the first strand synthesis reaction buffer later in the protocol, but it is important that it is ready before the elution in step 38. The beads should not be allowed to dry out.

Mix thoroughly by pipetting up and down 10 times.

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

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 (use magnet NEB #S1506 for 1.5 ml tubes). The purpose of this step is to bring the beads from the storage buffer into the binding buffer. The 2X Binding Buffer does not have to be diluted for this step.

A	B
Component	Volume Per One Library
Oligo dT Beads d(T)25	20 µl
RNA Binding Buffer	100 µl
Total Volume	120 µl

Wash the beads by pipetting up and down 6 times.

Place the tube on the magnet and incubate at room temperature until the solution is clear (~0h 2m 0s).

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

Remove the tube from the magnetic rack.

Add 100µL RNA Binding Buffer (2X) to the beads and wash by pipetting up and down six times. If preparing multiple libraries, add 100µL RNA Binding Buffer (2X) per sample. The Binding Buffer does not have to be diluted.

Place the tubes on the magnet and incubate at room temperature until the solution is clear (~0h 2m 0s).

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

Add50µL RNA Binding Buffer (2X) to the beads and mix by pipetting up and down until beads are homogenous. If preparing multiple libraries, add 50µL RNA Binding Buffer (2X) per sample. This first binding step removes most of the non target RNA.

Add 50 μl beads to each RNA sample from Step 3. Mix thoroughly by pipetting up and down 6 times.

Place the tube in a thermocycler and close the lid. Heat the sample at for and cool to with the heated lid set at ≥ 65°C for 0h 5m 0s 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.

Remove the tube from the thermocycler when the temperature reaches 4°C.

Mix thoroughly by pipetting up and down 6 times. Place the tube on the bench and incubate at room temperature for 0h 5m 0s to allow the mRNA to bind to the beads.

Place the tube on the magnetic rack at room temperature until the solution is clear (~0h 2m 0s).

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

Remove the tube from the magnetic rack.

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

Place the tube on the magnetic rack at room temperature until the solution is clear (~0h 2m 0s).

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

Remove the tube from the magnetic rack.

Repeat steps 20–23.

Add 50 μl of Tris Buffer (provided in NEB #E7490 kit) to each tube. Gently pipette up and down 6 times to mix thoroughly.

Place the tube on the thermocycler. Close the lid and heat the samples at80°C for , then cool to with the heated lid set at ≥ 0h 2m 0s, then cool to 25°C with the heated lid set at ≥ 90°C to do the first elution of the mRNA from the beads.

Remove the tube from the thermocycler when the temperature reaches 25°C.

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 six times.

Incubate the tube at room temperature for 0h 5m 0s.

Place the tube on the magnetic rack at room temperature until the solution is clear (~0h 2m 0s).

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

Remove the tube from the magnetic rack.

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

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

Place the tube on the magnet at room temperature until the solution is clear (~0h 2m 0s).

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

Remove the tube from the magnetic rack.

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

Incubate the sample in a thermocycler with the heated lid set at 105°C as follows:

at 0h 15m 0s at 94°C

*Hold at *** 4°C

*Immediately transfer the tube to ice for 0h 1m 0s as soon as it is cool enough to handle (~65°C)

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).

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

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

Assemble the first strand synthesis reaction on ice by adding the following components to the fragmented and primed RNA from Step 42:

A	B
First Strand Synthesis Reaction	Volume
Fragmented and Primed RNA (Step 36)	10 µl
NEBNext Strand Specificity Reagent	8 µl
NEBNext First Strand Synthesis Enzyme Mix	2 µl
Total Volume	20 µl

Mix thoroughly by pipetting up and down 10 times.

[ ! ] Incubate the sample in a preheated thermocycler with the heated lid set at ≥ 80°C as follows:

Note: If you are following recommendations in Appendix A (Chapter 6), for libraries with longer inserts (> 200 bases), increase the incubation at from 15 minutes to 50 minutes at Step 2 below. 42°C from 15 minutes to 50 minutes at Step 2 below.

Step 1: 0h 10m 0s at 25°C

Step 2: 0h 15m 0s at 42°C

Step 3: 0h 15m 0s at 70°C

Step 4: Hold at 4°C

Proceed directly to Second Strand cDNA Synthesis.

Assemble the second strand cDNA synthesis reaction on ice by adding the following components into the first strand synthesis product from Step 46).

A	B
Second Strand Synthesis Reaction	Volume
First Strand Synthesis Product (Step 46)	20 µl
NEBNext Second Strand Synthesis Reaction Buffer with dUTP (10X)	8 µl
NEBNext Second Strand Synthesis Enzyme Mix	4 µl
Nuclease-free Water	48 µl
Total Volume	80 µl

Keeping the tube on ice, mix thoroughly by pipetting up and down at least 10 times.

Incubate in a thermocycler for at 1h 0m 0s at 16°C with the heated lid set at 40°C (or off).

Vortex SPRIselect Beads or NEBNext Sample Purification Beads to resuspend.

Add 144µL (1.8X) of resuspended beads to the second strand synthesis reaction (~80µL). Mix well on a vortex mixer or by pipetting up and down at least 10 times.

Incubate for 0h 5m 0s at room temperature.

Briefly spin the tube in a microcentrifuge to collect any sample on the sides of the tube. Place the tube on a magnet 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.

Caution: Do not discard beads.

Add 200µL of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for 0h 0m 30s, and then carefully remove and discard the supernatant.

Repeat Step 54 once for a total of 2 washing steps.

Air dry the beads for up to 5 minutes while the tube is on the magnetic rack with lid open.

Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.

Remove the tube from the magnetic rack. Elute the DNA from the beads by adding 53µL 0.1X TE Buffer (provided) to the beads. Mix well on a vortex mixer or by pipetting up and down at least 10 times. Quickly spin the tube and incubate for 0h 2m 0s at room temperature. Place the tube on the magnetic rack until the solution is clear.

Remove 50µL of the supernatant and transfer to a clean nuclease-free PCR tube.

Assemble the end prep reaction on ice by adding the following components to the second strand synthesis product from Step 58.

A	B
End Prep Reaction	Volume
Second Strand Synthesis Product (Step 58)	50 µl
NEBNext Ultra II End Prep Reaction Buffer	7 µl
NEBNext Ultra II End Prep Enzyme Mix	3 µl
Total Volume	60 µl

If a master mix is made, add 10µL of master mix to 50µL of cDNA for the End Prep reaction.

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.

Incubate the sample in a thermocycler with the heated lid set at ≥ 75°Cas follows.

0h 30m 0s at 20°C

0h 30m 0s at 65°C

Hold at4°C

Proceed immediately to Adaptor Ligation.

[ ! ] Dilute the NEBNext Adaptor* prior to setting up the ligation reaction in ice-cold Adaptor Dilution Buffer and keep the adaptor on ice.

A	B
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 NEBNext adaptor is provided in NEBNext oligos kit. NEB has several oligo kit options, which are supplied separately from the library prep kit.

Assemble the ligation reaction on ice by adding the following components, in the order given, to the end prep reaction product from Step 62.

A	B
Ligation Reaction	Volume
End Prepped DNA (Step 62)	60 µl
Diluted Adaptor (Step 63)	2.5 µl
NEBNext Ligation Enhancer	1 µl
NEBNext Ultra II Ligation Master Mix	30 µl
Total Volume	93.5 µl

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.

Incubate 15 minutes at 20°C in a thermocycler.

0h 15m 0s

Add 3µL (blue) USER™ Enzyme to the ligation mixture from Step 60, resulting in total volume of 96.5µL

Mix well and incubate at for 37°C for 0h 15m 0swith the heated lid set to ≥45°C.

Proceed immediately to Purification of the Ligation Reaction.

[ ! ] Note: If you are selecting for libraries with larger insert size (> 200 nt) follow the size selection recommendations in Appendix A, Chapter 6.

Add 87µL (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.

Incubate for 0h 10m 0s at room temperature.

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 (~ 0h 5m 0s), discard the supernatant that contains unwanted fragments.

Caution: Do not discard beads.

Add 200µL of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for 0h 0m 30s, and then carefully remove and discard the supernatant.

Repeat Step 73 once for a total of 2 washing steps.

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

Completely remove the residual ethanol, and air dry beads until the beads are dry for up to 5 minutes while the tube is on the magnetic rack with the lid open.

[ ! ] Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.

Remove the tube from the magnetic rack. Elute DNA target from the beads by adding 17µL 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 0h 2m 0s at room temperature. Put the tube in the magnet until the solution is clear.

Without disturbing the bead pellet, transfer 15µL of the supernatant to a clean PCR tube and proceed to PCR enrichment.

[ ! ] Check and verify that the concentration of your oligos is 10 μM on the label.

[ ! ] Use Option A for any NEBNext oligos kit where index primers are supplied in tubes. These kits have the forward and reverse primers supplied in separate tubes.

Use Option B for any NEBNext oligos kit where index primers are supplied in a 96-well plate format. These kits have the forward and reverse (i7 and i5) primers combined.

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

Option A: Forward and Reverse Primers Separate:

A	B
Component	Volume Per One Library
Adaptor Ligated DNA (Step 78)	15 µl
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

Option B: Forward and Reverse Primers Combined:

A	B
Component	Volume Per One Library
Adaptor ligated DNA (Step 78)	15 µl
NEBNext Ultra II Q5 Master Mix	25 µl
Index (X)/Universal Primer Mix*	10 µl
Total Volume	50 µl

• NEBNext Oligos must be purchased separately from the library prep kit. Refer to the corresponding NEBNext Oligo kit manual for determining valid barcode combinations.

** Use only one i7 primer/ index primer per sample. Use only one i5 primer (or the universal primer for single index kits) per sample

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

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 82.A and Table 82.B):

Table 82.A:

A	B	C	D
Cycle Step	Temp	Time	Cycles
Initial Denaturation	98°C	30 seconds	1
Denaturation	98°C	10 seconds	8–16*,**
Annealing/Extension	65°C	15 seconds
Extension	72°C	20 seconds
Final Extension	72°C	5 minutes	1
Hold	4°C	∞

• The number of PCR cycles should be adjusted based on RNA input.

** 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 (See Figure 7.2 in Manual).

Table 82.B: Recommended PCR cycles based on total RNA input amount:

A	B
Total RNA Input	Recommended PCR Cycles
1,000 ng	8–9
100 ng	12–13
10 ng	15–16

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.

Vortex SPRIselect Beads or NEBNext Sample Purification Beads to resuspend.

Add 45µL (0.9X) of resuspended beads to the PCR reaction (~50µL). Mix well on a vortex mixer or by pipetting up and down at least 10 times.

Incubate for 0h 5m 0s at room temperature.

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 (~0h 5m 0s), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

Caution: Do not discard beads.

Add 200µL of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for 0h 0m 30s, and then carefully remove and discard the supernatant.

Repeat once for a total of 2 washing steps.

Air dry the beads for up to 5 minutes while the tube is on the magnetic rack with the lid open.

Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.

Remove the tube from the magnetic rack. Elute the DNA target from the beads by adding 23µL 0.1X TE (provided) to the beads. Mix well on a vortex mixer or by pipetting up and down ten times. Quickly spin the tube in a microcentrifuge and incubate for 0h 2m 0s at room temperature. Place the tube in the magnetic rack until the solution is clear.

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

Run 1µL 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. A dilution may be necessary for running on a Bioanalyzer High Sensitivity DNA Chip.

Check that the electropherogram shows a narrow distribution with a peak size approximately 300 bp.

Figure 94: Example of RNA library size distribution on a Bioanalyzer.