Aug 26, 2024
Protocol for use with NEBNext ULTRAEXPRESS® RNA Library Prep Kit (NEB#E3330) and NEBNext rRNA Depletion Kits (NEB #E7400, #E7405) and non indexed adaptor
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
Protocol Citation: Juliet Bonnevie 2024. Protocol for use with NEBNext ULTRAEXPRESS® RNA Library Prep Kit (NEB#E3330) and NEBNext rRNA Depletion Kits (NEB #E7400, #E7405) and non indexed adaptor. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw14o2lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 13, 2024
Last Modified: August 26, 2024
Protocol Integer ID: 106286
Keywords: RNA Library Prep Kit, PCR cycling
Abstract
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.
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
Materials
The Library Prep Kit Includes
The volumes provided are sufficient for preparation of up to 24 reactions (NEB #E3330S) and 96 reactions (NEB #E3330L)
Package 1: Store at -20 °C .
- (lilac) NEBNext UltraExpress RNA Fragmentation Mix (Protocol for use with NEBNext UltraExpress RNA Library Prep Kit (Neb #E3330), NEBNext rRNA Depletion Kits (NEB #E7400, #E7405) and NEBNext non-indexed adaptor)
- (lilac) NEBNext UltraExpress First Strand Enzyme Mix (Protocol for use with NEBNext UltraExpress RNA Library Prep Kit (Neb #E3330), NEBNext rRNA Depletion Kits (NEB #E7400, #E7405) and NEBNext non-indexed adaptor)
- (brown) NEBNext UltraExpress Strand Specificity Reagent (Protocol for use with NEBNext UltraExpress RNA Library Prep Kit (Neb #E3330), NEBNext rRNA Depletion Kits (NEB #E7400, #E7405) and NEBNext non-indexed adaptor)
- (orange) NEBNext UltraExpress Second Strand Master Mix (Protocol for use with NEBNext UltraExpress RNA Library Prep Kit (Neb #E3330), NEBNext rRNA Depletion Kits (NEB #E7400, #E7405) and NEBNext non-indexed adaptor)
- (green) NEBNext UltraExpress End Prep Enzyme Mix (Protocol for use with NEBNext UltraExpress RNA Library Prep Kit (Neb #E3330), NEBNext rRNA Depletion Kits (NEB #E7400, #E7405) and NEBNext non-indexed adaptor)
- (green) NEBNext UltraExpress End Prep Reaction Buffer (Protocol for use with NEBNext UltraExpress RNA Library Prep Kit (Neb #E3330), NEBNext rRNA Depletion Kits (NEB #E7400, #E7405) and NEBNext non-indexed adaptor)
- (red) NEBNext UltraExpress Ligation Master Mix (Protocol for use with NEBNext UltraExpress RNA Library Prep Kit (Neb #E3330), NEBNext rRNA Depletion Kits (NEB #E7400, #E7405) and NEBNext non-indexed adaptor)
- (blue) NEBNext UltraExpress USER‱ Enzyme (Protocol for use with NEBNext UltraExpress RNA Library Prep Kit (Neb #E3330), NEBNext rRNA Depletion Kits (NEB #E7400, #E7405) and NEBNext non-indexed adaptor)
- (blue) NEBNext MSTC High Yield Master Mix (Protocol for use with NEBNext UltraExpress RNA Library Prep Kit (Neb #E3330), NEBNext rRNA Depletion Kits (NEB #E7400, #E7405) and NEBNext non-indexed adaptor)
- (white) NEBNext Adaptor Dilution Buffer (Protocol for use with NEBNext UltraExpress RNA Library Prep Kit (Neb #E3330), NEBNext rRNA Depletion Kits (NEB #E7400, #E7405) and NEBNext non-indexed adaptor)
- (white) 0.1X TE (Protocol for use with NEBNext UltraExpress RNA Library Prep Kit (Neb #E3330), NEBNext rRNA Depletion Kits (NEB #E7400, #E7405) and NEBNext non-indexed adaptor)
- (white) Nuclease-free Water (Protocol for use with NEBNext UltraExpress RNA Library Prep Kit (Neb #E3330), NEBNext rRNA Depletion Kits (NEB #E7400, #E7405) and NEBNext non-indexed adaptor)
- (white) NEBNext Bead Reconstitution Buffer (Protocol for use with NEBNext UltraExpress RNA Library Prep Kit (Neb #E3330), NEBNext rRNA Depletion Kits (NEB #E7400, #E7405) and NEBNext non-indexed adaptor)
Required Materials Not Included
- NEBNext Multiplex Oligos for Illumina®
- NEBNext Multiplex Oligos options can be found at www.neb.com/oligos. Alternatively, customer supplied adaptor and primers can be used; please see information in link below: https://www.neb.com/faqs/2019/03/08/can-i-use-this-nebnext-kit-with-adaptors-and-primers-from-other-vendors-than-neb
- SPRIselect™ Reagent Kit (Beckman Coulter®, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881)
- Magnetic Rack (NEB S1515S, Alpaqua® cat. #A001322, or equivalent)
- 80% Ethanol (freshly prepared)
- Thermal cycler
- DNase-, RNase-free PCR strip tubes, for example TempAssure® PCR flex-free 8-tube strips (USA Scientific® #1402-4708)
- Bioanalyzer® or TapeStation® (Agilent® Technologies, Inc.) and associated reagents and consumables
For use with NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB #E7400) and other NEBNext RNA depletion kits that do not include beads (NEB #E7750, E7850, E7865):
- Agencourt® RNAClean® XP Beads (Beckman Coulter, Inc. #A63987)
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
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)
Preparation of 1X Fragmentation Mix for RNA elution
Preparation of 1X Fragmentation Mix for RNA elution
Thaw the Fragmentation Master Mix (2X) and prepare 1X composition as follows:
| A | B | |
| COMPONENT | VOLUME | |
| • (lilac) NEBNext UltraExpress RNA Fragmentation Mix | 4 μl | |
| Nuclease-free Water | 4 μl |
Note
Keep the mix On ice until RNA is purified. It will be used in Step 26.
Probe Hybridization to RNA
Probe Hybridization to RNA
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 .
Assemble the following RNA/Probe hybridization reaction On ice :
| A | B | |
| RNA/PROBE HYBRIDIZATION REACTION | VOLUME | |
| Total RNA in Nuclease-free Water (25–250 ng) | 11 μl | |
| • (white) NEBNext v2 rRNA Depletion Solution | 2 μl | |
| • (white) NEBNext Probe Hybridization Buffer | 2 μl | |
| Total Volume | 15 μl |
Mix thoroughly by pipetting up and down at least 10 times.
Note
It is crucial to mix well at this step.
Briefly spin down the tube in a microcentrifuge to collect the liquid from the side of the tube.
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.
| A | B | |
| TEMPERATURE | TIME | |
| 95°C | 2 minutes | |
| Ramp Down to 22°C | 0.1°C/sec | |
| Hold at 22°C | 5 minutes |
Briefly spin down the tube in a microcentrifuge and place On ice . Proceed immediately to the RNase H Digestion.
RNase H Digestion
RNase H Digestion
30m
30m
Assemble the following RNase H digestion reaction On ice :
| A | B | |
| RNASE H DIGESTION REACTION | VOLUME | |
| Hybridized RNA (Step 7) | 15 μl | |
| • (white) RNase H Reaction Buffer | 2 μl | |
| • (white) NEBNext Thermostable RNase H | 2 μl | |
| Nuclease-free Water | 1 μl | |
| Total Volume | 20 μl |
Mix thoroughly by pipetting up and down at least 10 times.
Briefly spin down the tube in a microcentrifuge.
Place in a pre-heated thermal cycler, with the heated lid set to 55 °C , and run the following program:
00:30:00 at 50 °C .
Hold at 4 °C .
30m
Briefly spin down the tube in a microcentrifuge and place On ice . Proceed immediately to DNase I Digestion.
DNase I Digestion
DNase I Digestion
30m
30m
Assemble the following DNase I digestion reaction On ice :
| A | B | |
| DNASE I DIGESTION REACTION | VOLUME | |
| RNase H treated RNA (Step 12) | 20 μl | |
| • (white) DNase I Reaction Buffer | 5 μl | |
| • (white) NEBNext DNase I (RNase-free) | 2.5 μl | |
| Nuclease-free Water | 22.5 μl | |
| Total Volume | 50 μl |
Mix thoroughly by pipetting up and down at least 10 times.
Briefly spin down the tube in a microcentrifuge.
Place in a thermal cycler, with the heated lid set at ≥ 45 °C , and run the following program:
00:30:00 at 37 °C
Hold at 4 °C
30m
Briefly spin down the tube in a microcentrifuge and place On ice . Proceed immediately to RNA Purification.
RNA Purification using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads
RNA Purification using Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads
38m
38m
Vortex the Agencourt RNAClean XP Beads or NEBNext RNA Sample Purification Beads to resuspend.
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.
Incubate for 00:15:00 On ice to bind RNA to the beads.
15m
Place the tube on a magnetic rack to separate the beads from the supernatant.
After the solution is clear, carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the RNA.
Add 200 µL of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at Room temperature for 00:00:30 and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the RNA.
30s
Repeat the previous step once for a total of 2 washes.
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.
Note
Caution: Do not over-dry the beads. This may result in lower recovery of RNA 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. Resuspend the RNA from the beads by adding 6.5 µL of Fragmentation Master Mix (1X). Mix thoroughly by pipetting up and down at least 10 times and briefly spin the tube. Incubate samples for 00:02:00 on bench top following elution and proceed to on-bead fragmentation.
Note
The next step provides a fragmentation incubation time resulting in an RNA insert size of ~ 200 nt. For RNA insert sizes > 200 nt, see Appendix D (in the library prep kit manual on NEB.com)
2m
Place in a thermal cycler, with the heated lid set at 105 °C , and run the following program:
00:15:00 at 94 °C
Hold at 4 °C
15m
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 RNA by transferring 5 µL of the supernatant to a nuclease-free 0.2 ml PCR tube.
Note
- If the supernatant volume recovered is less than 5 µL for any reason, bring the volume up to 5 µL by adding additional 1X Fragmentation Master Mix and continue with the protocol.
- Avoid transferring any of the magnetic beads.
Immediately transfer the tube to ice and proceed to First Strand cDNA Synthesis.
First Strand cDNA Synthesis
First Strand cDNA Synthesis
25m
25m
Assemble the first strand synthesis reaction On ice by adding the following components to the fragmented and primed RNA from Step 30:
| A | B | |
| FIRST STRAND SYNTHESIS REACTION | VOLUME | |
| Fragmented and Primed RNA (Step 30) | 5 μl | |
| • (brown) NEBNext UltraExpress Strand Specificity Reagent | 4 μl | |
| • (lilac) NEBNext UltraExpress First Strand Enzyme Mix | 1 μl | |
| Total Volume | 10 μl |
If processing multiple samples, prepare a master mix.
Mix thoroughly by pipetting up and down 10 times.
Place in a thermocycler, with the heated lid set at ≥ 80 °C , and run the following program:
00:10:00 at 25 °C
00:10:00 at 42 °C
00:05:00 at 70 °C
Hold at 4 °C
Proceed directly to Second Strand cDNA Synthesis.
25m
Second Strand cDNA Synthesis
Second Strand cDNA Synthesis
43m
43m
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.
| A | B | |
| SECOND STRAND SYNTHESIS REACTION | VOLUME | |
| First-Strand Synthesis Product (Step 34) | 10 μl | |
| • (orange) NEBNext UltraExpress Second Strand Master Mix | 30 μl | |
| Total Volume | 40 μl |
Keeping the tube On ice , mix thoroughly by pipetting the reaction up and down at least 10 times.
Place in a thermal cycler, with the heated lid set at ≤ 40 °C (or off), and run the following program:
00:30:00 at 16 °C
Hold at 4 °C
30m
Purification of double-stranded cDNA using SPRIselect Beads or AMPure Beads.
Purification of double-stranded cDNA using SPRIselect Beads or AMPure Beads.
Note
If using AMPure Beads, remove from 4 °C and keep at Room temperature for 30 minutes prior to use.
Vortex SPRIselect Beads or AMPure Beads to resuspend.
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.
Incubate samples on bench top for at least 00:05:00 at Room temperature .
5m
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.
Note
Caution: do not discard beads.
Add 200 µL of freshly prepared 80% ethanol to the tube while in the magnetic stand. Incubate at Room temperature for 00:00:30 , and then carefully remove and discard the supernatant.
30s
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.
Air dry the beads for up to 5 minutes while the tube is on the magnetic rack with the lid open.
Note
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/plate from the magnetic stand. Elute the DNA target from the beads by adding 22 µL 0.1X TE Buffer (provided) to the beads.
Mix well on a vortex mixer or by pipetting up and down 10 times. Incubate for at least 00:02:00 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.
2m
Place the tube/plate on a magnetic stand. After 00:05:00 (or when the solution is clear), transfer 20 µL to a new PCR tube.
Note
Safe Stop: If you need to stop at this point in the protocol, samples can be stored at -20 °C .
5m
End Prep of cDNA Library
End Prep of cDNA Library
15m
15m
Thaw the end prep master mix On ice . Add the following components to second strand synthesis product from Step 47.
| A | B | |
| END PREP REACTION | VOLUME | |
| Second Strand cDNA Synthesis Product (Step 47) | 20 μl | |
| • (green) NEBNext UltraExpress End Prep Reaction Buffer | 2.5 μl | |
| • (green) NEBNext UltraExpress End Prep Enzyme Mix | 1.5 μl |
If processing multiple samples, prepare a master mix.
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.
Note
It is important to mix well. The presence of a small amount of bubbles will not interfere with performance.
Place in a thermal cycler, with the heated lid set at ≥ 75 °C , and run the following program:
00:05:00 at 20 °C
00:10:00 at 65 °C
Hold at 4 °C
Proceed immediately to Adaptor Ligation.
15m
Adaptor Ligation
Adaptor Ligation
20m
20m
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 .
| A | B | |
| TOTAL RNA INPUT | DILUTION REQUIRED | |
| 25–250 ng | 50-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.
Note
If customized adaptor titration per input is preferred use recommendations in Table 3.1.1. Appendix A (in the library prep kit manual on NEB.com)
Assemble the ligation reaction On ice by adding the following components, in the order given, to the end prep reaction product from Step 51.
| A | B | |
| LIGATION REACTION | VOLUME | |
| End Prepped DNA (Step 51) | 24 μl | |
| Diluted Adaptor (Step 52) | 2 μl | |
| • (red) NEBNext UltraExpress Ligation Master Mix | 12 μl | |
| Total Volume | 38 μl |
Note
Do not premix the Ligation Master Mix and adaptor prior to use in the Adaptor Ligation Step.
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.
Note
Caution: NEBNext UltraExpress 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.
Place in a thermal cycler, with the heated lid set at ≤ 40 °C (or off), and run the following program:
00:15:00 at 20 °C
Hold at 4 °C
15m
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.
Place in a thermal cycler, with the heated lid set at ≥ 45 °C , and run the following program:
37 °C for 00:05:00
Hold at 4 °C
Proceed immediately to PCR Enrichment of Adaptor Ligated DNA.
5m
PCR Enrichment of Adaptor Ligated DNA
PCR Enrichment of Adaptor Ligated DNA
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).
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 57) | 40 μl | |
| • (blue) NEBNext MSTC High Yield Master Mix | 50 μl | |
| Index (X) Primer/i7 Primer*, ** | 5 μl | |
| Universal PCR Primer/i5 Primer*,** | 5 μl | |
| Total Volume | 100 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
Note
Skip the next step if you are choosing this step.
Option B:
Forward and Reverse Primers Combined
| A | B | |
| COMPONENT | VOLUME PER ONE LIBRARY | |
| Adaptor Ligated DNA (Step 57) | 40 μl | |
| • (blue) NEBNext MSTC High Yield Master Mix | 50 μl | |
| Index Primer Mix* | 10 μl | |
| Total Volume | 100 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.
Note
Skip 59.2 if choosing 59.1
Mix well by gently pipetting up and down 10 times. Quickly spin the tube in a microcentrifuge.
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:
** PCR cycles are recommended based on high quality Universal Human Reference Total RNA. It may require optimization based on the sample quality. If using customized cycling conditions per input is preferred, use recommendations in Table 3.2.2 in Appendix A (in the library prep kit manual on NEB.com).
Note
Take out NEBNext Bead Reconstitution Buffer and bring up to Room temperature prior to Phased Bead Cleanup. Allow the buffer (and beads if using AMPure XP) to warm to Room temperature for at least 30 minutes before use.
Phased Bead Cleanup of PCR Reaction
Phased Bead Cleanup of PCR Reaction
5m
5m
Note
Note: The SPRIselect/AMPure Bead ratio recommended in this manual have been experimentally optimized for every step; this is critical since buffer compositions differ between steps and across protocols e.g. post ligation recommendations will not apply to samples post PCR. Please adhere to these guidelines and not those recommended by other sources or for other kits. If using AMPure Beads, remove from 4 °C and keep at Room temperature for 30 minutes prior to use.
Vortex SPRIselect Beads or AMPure Beads to resuspend.
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.
Incubate samples on bench top for at least 00:05:00 at Room temperature .
5m
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.
After 00:05:00 (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Note
Caution: do not discard the beads
5m
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.
Incubate samples on bench top for at least 00:05:00 at Room temperature .
5m
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.
After 00:05:00 (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Note
Caution: do not discard the beads
5m
Add 200 µL of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at Room temperature for 00:00:30 , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
30s
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.
Air dry the beads for up to 5 minutes while the tube is on the magnetic rack with the lid open.
Note
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/plate from the magnetic stand. Elute the DNA target from the beads by adding 23 µL 0.1X TE (provided) to the beads.
Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 00:02:00 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.
2m
Place the tube/plate on the magnetic stand. After 00:05:00 (or when the solution is clear), transfer 20 µL to a new PCR tube or plate and store at -20 °C .
5m
Assess Library Quality on an Agilent Bioanalyzer DNA Chip
Assess Library Quality on an Agilent Bioanalyzer DNA Chip
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.
Check that the electropherogram shows a narrow distribution with a peak size approximately 300 bp.
Note
If a peak at ~ 80 bp (primers) or 150 bp (adaptor-dimer) is visible in the traces, bring up the sample volume (from Step 77) to 50 µL with 0.1X TE buffer and repeat the SPRIselect Bead or NEBNext Sample Purification Bead Cleanup Step using 0.9X (45 μl) standard bead clean-up.
Figure 79. Example of library made using 250 ng of UHRR on a TapeStation.