TITLE: Protocol for use with NEBNext ULTRAEXPRESS® RNA Library Prep Kit (NEB#E3330) and NEBNext rRNA Depletion Kits (NEB #E7400, #E7405) and non indexed adaptor
AUTHORS: Juliet Bonnevie

[DESCRIPTION]
The NEBNext UltraExpress RNA Library Prep Kit contains the enzymes and buffers required to to rapidly convert 25–250 ng of total RNA into high-quality libraries for next-generation sequencing on the Illumina platform. The fast, simple workflow features minimal hands-on time and allows use of a single adaptor dilution and PCR cycling condition across the entire input range. In addition to the standard protocol, an appendix is included that details customized adaptor and cycling recommendations for varying RNA input amounts, if further optimization for library yields is required. If a stopping point is required between mRNA enrichment/ribosomal RNA depletion and library preparation, please refer to the FAQs tab for considerations and recommendations the FAQ tab is on NEB.com.


[BEFORE_START]
Considerations on Selecting Poly(A) mRNA Enrichment or rRNA Depletion

The library preparation protocol should be chosen based on the goals of the project and the quality of the RNA sample. Total cellular RNA is mainly composed of ribosomal RNA (rRNA) and often is not of interest. rRNA can be removed from total cellular RNA with either of two common methods. The first method uses oligo d(T) beads, which bind to the poly(A) tail of eukaryotic mRNA. Alternatively, rRNA can be depleted using rRNA-specific probes. NEB offers the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) and the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) with or without RNA Sample Purification Beads (NEB #E7400/ #E7405) for the enrichment of non-ribosomal RNA.

In the oligo d(T) approach, only mRNA with poly(A) tails will be enriched; other cellular RNA without a poly(A) tail, such as non-coding RNA or mRNA lacking poly(A) will not bind to the beads. In addition, mRNA from some organisms (e.g., prokaryotes) or degraded RNA (e.g., FFPE RNA) do not have poly(A) tails and will not be captured by oligo d(T) beads. On the other hand, the probe-based rRNA depletion kit will remove the targeted rRNA, but it will preserve other biologically relevant cellular RNA such as non-coding RNA or mRNA.

The quality of an RNA sample should also be considered when deciding on a library preparation protocol. The NEBNext Poly(A) mRNA Magnetic Isolation Module should only be used with high-quality RNA samples (RIN > 7), since degradation results in a loss of poly(A) tails from mRNA molecules. For partially degraded or heavily degraded samples (e.g., RIN ≤ 7, FFPE RNA), the NEBNext rRNA Depletion Kit should be used.

RNA Sample Requirements

RNA Integrity

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). For intact (RIN > 7) or partially degraded RNA samples (RIN = 2 to 7) follow the library preparation protocol in sections in the kit  manual on NEB.com. For highly degraded samples (e.g., FFPE) which do not require fragmentation, follow the library preparation protocol recommendations in appendix B (in the library prep kit manual on NEB.com)
RNA Purity

The RNA sample should be free of salts (e.g., Mg2+, or guanidinium salts) or organics (e.g., phenol and ethanol). RNA must be free of DNA. gDNA is a common contaminant from RNA preps. It may be carried over from the interphase of organic extractions or when the silica matrix of solid phase RNA purification methods is overloaded. If the total RNA sample may contain gDNA contamination, treat the sample with DNase I to remove all traces of DNA (not provided in this kit). After treatment with DNase I the enzyme should be removed from the sample. Any residual activity of DNase I will degrade the single stranded DNA probes necessary for the ribosomal depletion. DNase I can be removed from the extraction using phenol/ chloroform extraction and ethanol precipitation. Prior to depletion the RNA must be in nuclease free water. Some products, e.g., TURBO DNA-free™ Kit TURBO™ DNase Treatment and Removal Reagents do not produce RNA in nuclease free water and are not compatible with NEBNext rRNA depletion.

Input Amount

25–250 ng DNA-free total RNA quantified by Qubit Fluorometer or spectrophotometer and quality checked by Bioanalyzer. The protocol is optimized for approximately 200 bp RNA inserts.

Prior to Starting Enrichment

Remove the following components to thaw on ice:

• NEBNext UltraExpress RNA Fragmentation Mix
• NEBNext UltraExpress Second Strand Master Mix (Note: do not vortex reagent, mix only by inversion)

[GUIDELINES]
The FAQ tab is on NEB.com for additional information about this product.

For larger volume requirements, customized and bulk packaging is available by purchasing through the Customized Solutions Team at NEB. Please contact custom@neb.com for further information.

Figure:1 NEBNext UltraExpress RNA Workflow


 

 


[STEPS]
SECTION: Preparation of 1X Fragmentation Mix for RNA elution
1. Thaw the Fragmentation Master Mix (2X) and prepare 1X composition as follows:

 

 

SECTION: Probe Hybridization to RNA
2. Dilute 25 ng–250 ng of total RNA with Nuclease-free Water to a final volume of 11 µL in a PCR tube. Keep the RNA on ice.

SECTION: Probe Hybridization to RNA
3. Assemble the following RNA/Probe hybridization reaction on ice:
 

SECTION: RNase H Digestion
8. Assemble the following RNase H digestion reaction on ice:

 

SECTION: RNase H Digestion
9. Mix thoroughly by pipetting up and down at least 10 times.
SECTION: RNase H Digestion
10. Briefly spin down the tube in a microcentrifuge.
SECTION: RNase H Digestion
11. Place in a pre-heated thermal cycler, with the heated lid set to 55 °C, and run the following program:

30 min at 50 °C.
Hold at 4 °C.

SECTION: RNase H Digestion
12. Briefly spin down the tube in a microcentrifuge and place on ice. Proceed immediately to DNase I Digestion.

SECTION: DNase I Digestion
13. Assemble the following DNase I digestion reaction on ice:

 

SECTION: DNase I Digestion
14. Mix thoroughly by pipetting up and down at least 10 times.
SECTION: DNase I Digestion
15. Briefly spin down the tube in a microcentrifuge.
SECTION: DNase I Digestion
16. Place in a thermal cycler, with the heated lid set at ≥ 45 °C, and run the following program:

30 min at 37 °C
Hold at 4 °C

SECTION: DNase I Digestion
17. Briefly spin down the tube in a microcentrifuge and place on ice. Proceed immediately to RNA Purification.

SECTION: RNA Purification using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads
18. Vortex the Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads to resuspend.
SECTION: RNA Purification using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads
19. Add 90 µL (1.8X) beads to the RNA sample from Step 17 and mix thoroughly by pipetting up and down at least 10 times.

SECTION: RNA Purification using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads
20. Incubate for 15 min on ice to bind RNA to the beads.

SECTION: RNA Purification using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads
21. Place the tube on a magnetic rack to separate the beads from the supernatant.
SECTION: RNA Purification using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads
22. After the solution is clear, carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the RNA.
SECTION: RNA Purification using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads
23. Add 200 µL of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at Room temperature for 30 s and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the RNA.
SECTION: RNA Purification using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads
24. Repeat the previous step once for a total of 2 washes. 
SECTION: RNA Purification using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads
25. Completely remove residual ethanol and air dry the beads for up to 5 minutes while the tube is 
 on the magnetic rack with the lid open.

 


SECTION: RNA Purification using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads
26. Remove the tube from the magnetic rack. Resuspend the RNA from the beads by adding 6.5 µLof Fragmentation Master Mix (1X). Mix thoroughly by pipetting up and down at least 10 times and briefly spin the tube. Incubate samples for 2 min on bench top following elution and proceed to on-bead fragmentation.

 


SECTION: RNA Purification using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads
27. Place in a thermal cycler, with the heated lid set at 105 °C, and run the following program:
15 min at 94 °C
Hold at 4 °C

SECTION: RNA Purification using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads
28. 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).
SECTION: RNA Purification using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads
29. Collect the fragmented RNA by transferring 5 µL of the supernatant to a nuclease-free 0.2 ml PCR tube.

 

SECTION: RNA Purification using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads
30. Immediately transfer the tube to ice and proceed to First Strand cDNA Synthesis.
SECTION: First Strand cDNA Synthesis
31. Assemble the first strand synthesis reaction on ice by adding the following components to the fragmented and primed RNA from Step 30:

 

SECTION: First Strand cDNA Synthesis
32. If processing multiple samples, prepare a master mix.
SECTION: First Strand cDNA Synthesis
33. Mix thoroughly by pipetting up and down 10 times.
SECTION: First Strand cDNA Synthesis
34. Place in a thermocycler, with the heated lid set at ≥ 80 °C, and run the following program:
10 min at 25 °C
10 min at 42 °C
5 min at 70 °C
Hold at 4 °C
Proceed directly to Second Strand cDNA Synthesis.
SECTION: Second Strand cDNA Synthesis
35. Thaw the second strand master mix on ice and mix by inverting tube 10 times, perform a quick spin to collect all liquid from the sides of the tube and place back on ice. Add the following components into the first strand synthesis reaction product from Step 34.

 

SECTION: Second Strand cDNA Synthesis
36. Keeping the tube on ice, mix thoroughly by pipetting the reaction up and down at least 10 times.

SECTION: Second Strand cDNA Synthesis
37. Place in a thermal cycler, with the heated lid set at ≤ 40 °C (or off), and run the following program:

30 min at 16 °C
Hold at 4 °C


SECTION: Purification of double-stranded cDNA using SPRIselect Beads or AMPure Beads.
38. 
 
Vortex SPRIselect Beads or AMPure Beads to resuspend.
SECTION: Purification of double-stranded cDNA using SPRIselect Beads or AMPure Beads.
39. Add 72 µL (1.8X) of resuspended beads to the second strand synthesis reaction (~40 µL). Mix well by pipetting up and down at least 10 times. Be careful to expel all the liquid out of the tip during the last mix. Vortexing for 3–5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

SECTION: Purification of double-stranded cDNA using SPRIselect Beads or AMPure Beads.
40. Incubate samples on bench top for at least 5 min at Room temperature.

SECTION: Purification of double-stranded cDNA using SPRIselect Beads or AMPure Beads.
41. Briefly spin the tube in a microcentrifuge to collect any sample from 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. 
 

SECTION: Purification of double-stranded cDNA using SPRIselect Beads or AMPure Beads.
42. Add 200 µL of freshly prepared 80% ethanol to the tube while in the magnetic stand. Incubate at Room temperature for 30 s, and then carefully remove and discard the supernatant.

SECTION: Purification of double-stranded cDNA using SPRIselect Beads or AMPure Beads.
43. Repeat the previous step once for a total of 2 washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
SECTION: Purification of double-stranded cDNA using SPRIselect Beads or AMPure Beads.
44. Air dry the beads for up to 5 minutes while the tube is on the magnetic rack with the lid open.
 



SECTION: Purification of double-stranded cDNA using SPRIselect Beads or AMPure Beads.
45. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 22 µL 0.1X TE Buffer (provided) to the beads.

SECTION: Purification of double-stranded cDNA using SPRIselect Beads or AMPure Beads.
46. Mix well on a vortex mixer or by pipetting up and down 10 times. Incubate for at least 2 min at Room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

SECTION: Purification of double-stranded cDNA using SPRIselect Beads or AMPure Beads.
47. Place the tube/plate on a magnetic stand. After 5 min (or when the solution is clear), transfer 20 µL to a new PCR tube.
 


SECTION: End Prep of cDNA Library
48. Thaw the end prep master mix on ice. Add the following components to second strand synthesis product from Step 47.

 

SECTION: End Prep of cDNA Library
49. If processing multiple samples, prepare a master mix.
SECTION: End Prep of cDNA Library
50. Set a 20 μl or 100 μl pipette to 20 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.
 

SECTION: End Prep of cDNA Library
51. Place in a thermal cycler, with the heated lid set at ≥ 75 °C, and run the following program:

5 min at 20 °C
10 min at 65 °C
Hold at 4 °C

Proceed immediately to Adaptor Ligation.
SECTION: Adaptor Ligation
52. 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.
 
* The NEBNext adaptor is provided in NEBNext oligos kit. NEB has several oligo kit options, which are supplied separately from the library prep kit.
 

 

SECTION: Adaptor Ligation
53. Assemble the ligation reaction on ice by adding the following components, in the order given, to the end prep reaction product from Step 51.

 

 
 


SECTION: Adaptor Ligation
54. Set a 100 μl pipette to 35 μl and then pipette the entire volume up and down at moderate speed at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
 

SECTION: Adaptor Ligation
55. Place in a thermal cycler, with the heated lid set at ≤ 40 °C (or off), and run the following program:

15 min at 20 °C
Hold at 4 °C


SECTION: Adaptor Ligation
56. Add 2 µL (• blue) NEBNext UltraExpress USER Enzyme to the ligation mixture from Step 55, resulting in total volume of 40 µL. Mix well by gently pipetting up and down 10 times at 35 µL volume.
SECTION: Adaptor Ligation
57. Place in a thermal cycler, with the heated lid set at ≥ 45 °C, and run the following program:

37 °C for 5 min
Hold at 4 °C

Proceed immediately to PCR Enrichment of Adaptor Ligated DNA.
SECTION: PCR Enrichment of Adaptor Ligated DNA
58. 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 (i5and i7) primers combined. Primers are supplied at 10 μM combined (5 μM each). 
SECTION: PCR Enrichment of Adaptor Ligated DNA
59. Set up the PCR reaction as described below based on the type of oligos (PCR primers) used.
SECTION: PCR Enrichment of Adaptor Ligated DNA
59.1. Option A:

Forward and Reverse Primers Separate

 

 

SECTION: PCR Enrichment of Adaptor Ligated DNA
59.2. Option B:

Forward and Reverse Primers Combined

 

 

SECTION: PCR Enrichment of Adaptor Ligated DNA
60. Mix well by gently pipetting up and down 10 times. Quickly spin the tube in a microcentrifuge.
SECTION: PCR Enrichment of Adaptor Ligated DNA
61. Place the tube on a thermal cycler with the heated lid set to 105 °C and perform PCR amplification using the following PCR cycling conditions (refer to Table 61):

Table 61:

 

 

SECTION: Phased Bead Cleanup of PCR Reaction
62. 
 

SECTION: Phased Bead Cleanup of PCR Reaction
63. Vortex SPRIselect Beads or AMPure Beads to resuspend.
SECTION: Phased Bead Cleanup of PCR Reaction
64. Add 70 µL (0.7X) of resuspended beads to the PCR reaction (~100 µL). Mix well by pipetting up and down at least 10 times. Be careful to expel all the liquid out of the tip during the last mix. Vortexing for 3–5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

SECTION: Phased Bead Cleanup of PCR Reaction
65. Incubate samples on bench top for at least 5 min at Room temperature.

SECTION: Phased Bead Cleanup of PCR Reaction
66. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
SECTION: Phased Bead Cleanup of PCR Reaction
67. After 5 min (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
 



SECTION: Phased Bead Cleanup of PCR Reaction
68. Remove the tube/plate from the magnetic stand (Note: do not need ethanol wash at this step). Add 50 µL 0.1 X TE and resuspend the beads by pipetting. Add another 40 µL (0.8X) NEBNext Bead Reconstitution Buffer to the 50 µL resuspended beads. Mix well by pipetting up and down at least 10 times. Be careful to expel all the liquid out of the tip during the last mix. Vortex for 3–5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.



SECTION: Phased Bead Cleanup of PCR Reaction
69. Incubate samples on bench top for at least 5 min at Room temperature.

SECTION: Phased Bead Cleanup of PCR Reaction
70. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
SECTION: Phased Bead Cleanup of PCR Reaction
71. After 5 min (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
 



SECTION: Phased Bead Cleanup of PCR Reaction
72. Add 200 µL of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at Room temperature for 30 s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

SECTION: Phased Bead Cleanup of PCR Reaction
73. Repeat the previous step once for a total of 2 washes.
Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
SECTION: Phased Bead Cleanup of PCR Reaction
74. Air dry the beads for up to 5 minutes while the tube is on the magnetic rack with the lid open.

 

SECTION: Phased Bead Cleanup of PCR Reaction
75. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 23 µL 0.1X TE (provided) to the beads.

SECTION: Phased Bead Cleanup of PCR Reaction
76. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min at Room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

SECTION: Phased Bead Cleanup of PCR Reaction
77. Place the tube/plate on the magnetic stand. After 5 min (or when the solution is clear), transfer 20 µL to a new PCR tube or plate and store at -20 °C.

SECTION: Assess Library Quality on an Agilent Bioanalyzer DNA Chip
78. Run 1 µL 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.

SECTION: Assess Library Quality on an Agilent Bioanalyzer DNA Chip
79. Check that the electropherogram shows a narrow distribution with a peak size approximately 300 bp.

 

 

SECTION: Probe Hybridization to RNA
4. Mix thoroughly by pipetting up and down at least 10 times.
 

SECTION: Probe Hybridization to RNA
5. Briefly spin down the tube in a microcentrifuge to collect the liquid from the side of the tube.
SECTION: Probe Hybridization to RNA
6. Place tube in a pre-heated thermocycler and run the following program with the heated lid set to 105 °C. This will take approximately 15–20 minutes to complete. 
 
 
SECTION: Probe Hybridization to RNA
7. Briefly spin down the tube in a microcentrifuge and place on ice. Proceed immediately to the RNase H Digestion.