Dec 19, 2018
Version 1
Protocol for use with Purified mRNA or rRNA Depleted RNA and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760, E7765) V.1
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
Protocol Citation: New England Biolabs 2018. Protocol for use with Purified mRNA or rRNA Depleted RNA and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760, E7765). protocols.io https://dx.doi.org/10.17504/protocols.io.t8xerxn
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
Created: October 02, 2018
Last Modified: July 22, 2020
Protocol Integer ID: 16375
Abstract
The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina contains the enzymes and buffers required to convert a broad range of input amounts of RNA into high quality directional (strand-specific) libraries for next-generation sequencing on the Illumina platform. The fast, user-friendly workflow has minimal hands-on time and is compatible with poly(A) mRNA enrichment and rRNA depletion methods.
Guidelines
Section 4
RNA Sample Requirements
This Section can be used for libraries without any enrichment or depletion of total RNA with RIN scores > 7.
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. In this case, we recommend that the fragmentation time is empirically determined if the RNA sample is suspected to be low quality. 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 Section 4 (current Section). See Table 4.1.1 for the recommended the fragmentation times.
For highly degraded samples (RIN = 1 to 2) (e.g. FFPE), which do not require fragmentation, follow the library preparation protocol in Section 5.
RNA Sample Requirements:
The RNA sample should be free of salts (e.g. Mg2+, or guanidinium salts, divalent cation chelating agents (e.g. EDTA or EGTA) or organics (e.g. phenol or 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 (DNase is not provided in this kit). After treatment with DNase I the enzyme should be removed from the sample. DNase I can be removed from the extraction using phenol/chloroform extraction and ethanol precipitation.
Materials
MATERIALS
NEBNext RNase HNew England BiolabsCatalog #E6318
RNase H Reaction BufferNew England BiolabsCatalog #E6312
NEBNext rRNA Depletion SolutionNew England BiolabsCatalog #E6313
NEBNext Probe Hybridization BufferNew England BiolabsCatalog #E6314
DNase I (RNase-free)New England BiolabsCatalog #E6316
DNase I Reaction BufferNew England BiolabsCatalog #E6315
Nuclease-free WaterNew England BiolabsCatalog #E6317
NEBNext RNA Sample Purification BeadsNew England BiolabsCatalog #E6315
Magnetic Rack
80% Ethanol (freshly prepared)
Thermal cycler
Agencourt RNAClean XP BeadsBeckman CoulterCatalog #A63987
DNase I (e.g., NEB #M0303) and DNase I Cleanup Reagants or Kit for Removal of DNA Prior to DepletionNew England Biolabs
Random PrimersNew England BiolabsCatalog #E7422
STEP MATERIALS
NEBNext Strand Specificity ReagentNew England BiolabsCatalog #E7766
NEBNext First Strand Synthesis Enzyme MixNew England BiolabsCatalog #E7761
NEBNext Second Strand Synthesis Reaction Buffer with dUTP MixNew England BiolabsCatalog #E7426
NEBNext Second Strand Synthesis Enzyme MixNew England BiolabsCatalog #E7425
Nuclease-free WaterNew England BiolabsCatalog #E7764
NEBNext Sample Purification BeadsNew England BiolabsCatalog #E7767
Fresh 80% Ethanol
(0.1X) TE BufferNew England BiolabsCatalog #E7763
NEBNext Ultra II End Prep Reaction BufferNew England BiolabsCatalog #E7647
NEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646
NEBNext Ligation EnhancerNew England BiolabsCatalog #E7374
NEBNext Ultra II Ligation Master MixNew England BiolabsCatalog #E7648
NEBNext USER EnzymeNew England BiolabsCatalog #E7458
NEBNext Sample Purification BeadsNew England BiolabsCatalog #E7767
80% Ethanol (freshly prepared)
NEBNext Sample Purification BeadsNew England BiolabsCatalog #E6315
80% Ethanol (freshly prepared)
Protocol materials
Fresh 80% Ethanol
NEBNext RNA Sample Purification BeadsNew England BiolabsCatalog #E6315
Thermal cycler
NEBNext Ultra II Ligation Master MixNew England BiolabsCatalog #E7648
NEBNext USER EnzymeNew England BiolabsCatalog #E7458
80% Ethanol (freshly prepared)
NEBNext RNase HNew England BiolabsCatalog #E6318
DNase I (e.g., NEB #M0303) and DNase I Cleanup Reagants or Kit for Removal of DNA Prior to DepletionNew England Biolabs
NEBNext First Strand Synthesis Enzyme MixNew England BiolabsCatalog #E7761
NEBNext Second Strand Synthesis Reaction Buffer with dUTP MixNew England BiolabsCatalog #E7426
NEBNext Sample Purification BeadsNew England BiolabsCatalog #E7767
(0.1X) TE BufferNew England BiolabsCatalog #E7763
NEBNext Ultra II End Prep Reaction BufferNew England BiolabsCatalog #E7647
NEBNext Sample Purification BeadsNew England BiolabsCatalog #E7767
Random PrimersNew England BiolabsCatalog #E7422
NEBNext Second Strand Synthesis Enzyme MixNew England BiolabsCatalog #E7425
NEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646
80% Ethanol (freshly prepared)
Nuclease-free WaterNew England BiolabsCatalog #E6317
RNase H Reaction BufferNew England BiolabsCatalog #E6312
NEBNext Strand Specificity ReagentNew England BiolabsCatalog #E7766
DNase I (RNase-free)New England BiolabsCatalog #E6316
NEBNext Probe Hybridization BufferNew England BiolabsCatalog #E6314
Magnetic Rack
Agencourt RNAClean XP BeadsBeckman CoulterCatalog #A63987
Nuclease-free WaterNew England BiolabsCatalog #E7764
NEBNext rRNA Depletion SolutionNew England BiolabsCatalog #E6313
NEBNext Ligation EnhancerNew England BiolabsCatalog #E7374
NEBNext Sample Purification BeadsNew England BiolabsCatalog #E6315
DNase I Reaction BufferNew England BiolabsCatalog #E6315
80% Ethanol (freshly prepared)
NEBNext Strand Specificity ReagentNew England BiolabsCatalog #E7766
NEBNext First Strand Synthesis Enzyme MixNew England BiolabsCatalog #E7761
NEBNext Second Strand Synthesis Enzyme MixNew England BiolabsCatalog #E7425
Nuclease-free WaterNew England BiolabsCatalog #E7764
NEBNext Second Strand Synthesis Reaction Buffer with dUTP MixNew England BiolabsCatalog #E7426
NEBNext Sample Purification BeadsNew England BiolabsCatalog #E7767
Fresh 80% Ethanol
(0.1X) TE BufferNew England BiolabsCatalog #E7763
NEBNext Ultra II End Prep Reaction BufferNew England BiolabsCatalog #E7647
NEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646
NEBNext Ligation EnhancerNew England BiolabsCatalog #E7374
NEBNext Ultra II Ligation Master MixNew England BiolabsCatalog #E7648
NEBNext USER EnzymeNew England BiolabsCatalog #E7458
NEBNext Sample Purification BeadsNew England BiolabsCatalog #E7767
80% Ethanol (freshly prepared)
NEBNext Sample Purification BeadsNew England BiolabsCatalog #E6315
80% Ethanol (freshly prepared)
Safety warnings
Please refer to the SDS (Safety Data Sheet) for safety warnings and hazard information.
Before start
Input Amount Requirements
1 ng – 100 ng purified mRNA or rRNA depleted RNA that is quantified after the purification. RNA should be DNA free in up to 5 μl of Nuclease-free Water, quantified by Qubit Fluorometer and quality checked by Bioanalyzer.
The protocol is optimized for approximately 200 nt RNA inserts. To generate libraries with longer RNA insert sizes, refer to Appendix A, (Section 6) for recommended fragmentation times and size selection conditions.
This protocol has been optimized using Universal Human Reference Total RNA.
RNA Fragmentation and Priming
RNA Fragmentation and Priming
[ ! ] RNA fragmentation is only required for intact or partially degraded RNA. Recommended fragmentation times can be found in Table 1 (step 3).
Assemble the fragmentation and priming reaction on ice in a nuclease-free tube by adding the following components:
| Fragmentation and Priming Mix | Volume | |
| Purified mRNA or rRNA Depleted RNA | 5 µl | |
| NEBNext First Strand Synthesis Reaction Buffer | 4 µl | |
| Random Primers | 1 µl | |
| Total Volume | 10 µl |
Mix thoroughly by pipetting up and down 10 times.
Place the sample in a thermocycler and incubate the sample at 94 °C following the recommendations in Table 1 below for fragment sizes ~200 nt.
Table 1. Suggested fragmentation times based on RIN value of RNA input.
| RNA Type | RIN | Frag. Time | |
| Intact RNA | > 7 | 15 min @ 94°C | |
| Partially Degraded RNA | 2–6 | 7–8 min @ 94°C |
Note
Refer to Appendix A, (Chapter 6) for fragmentation conditions if you are preparing libraries with large inserts (> 200 bp). Conditions in Appendix A, (Chapter 6) only apply for intact RNA.
Immediately transfer the tube to ice and proceed to First Strand cDNA Synthesis.
First Strand cDNA Synthesis
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 4:
| First Strand Synthesis Reaction | Volume | |
| Fragmented and Primed RNA (Step 4) | 10 µl | |
| NEBNext Strand Specificity Reagent | 8 µl | |
| NEBNext First Strand Synthesis Enzyme Mix | 2 µl | |
| Total Volume | 20 µl |
NEBNext Strand Specificity ReagentNew England BiolabsCatalog #E7766
NEBNext First Strand Synthesis Enzyme MixNew England BiolabsCatalog #E7761
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 42°C from 15 minutes to 50 minutes at Step 2 below.
Step 1: 00:10:00 at 25 °C
Step 2: 00:15:00 at 42 °C
Step 3: 00:15:00 at 70 °C
Step 4: Hold at 4 °C
Proceed directly to Second Strand cDNA Synthesis.
Second Strand cDNA Synthesis
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 8).
| Second Strand Synthesis Reaction | Volume | |
| First Strand Synthesis Product (Step 8) | 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 |
NEBNext Second Strand Synthesis Reaction Buffer with dUTP MixNew England BiolabsCatalog #E7426
NEBNext Second Strand Synthesis Enzyme MixNew England BiolabsCatalog #E7425
Nuclease-free WaterNew England BiolabsCatalog #E7764
Keeping the tube on ice, mix thoroughly by pipetting up and down at least 10 times.
Incubate in a thermocycler for 01:00:00 at 16 °C with the heated lid set at ≤ 40 °C (or off).
Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads
Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads
Vortex SPRIselect Beads or NEBNext Sample Purification Beads to resuspend.
NEBNext Sample Purification BeadsNew England BiolabsCatalog #E7767
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.
Incubate for 00:05:00 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 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.
Fresh 80% Ethanol
Repeat Step 16 once for a total of 2 washing steps.
go to step #16 Repeat Step
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 53 µL 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 00:02:00 at room temperature. Place the tube on the magnetic rack until the solution is clear.
(0.1X) TE BufferNew England BiolabsCatalog #E7763
Remove 50 µL of the supernatant and transfer to a clean nuclease-free PCR tube.
Note
If you need to stop at this point in the protocol samples can be stored at –20°C.
End Prep of cDNA Library
End Prep of cDNA Library
Assemble the end prep reaction on ice by adding the following components to the second strand synthesis product from Step 20.
| End Prep Reaction | Volume | |
| Second Strand Synthesis Product (Step 20) | 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 0 µL of master mix to 50 µL of cDNA for the End Prep reaction.
NEBNext Ultra II End Prep Reaction BufferNew England BiolabsCatalog #E7647
NEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646
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.
Incubate the sample in a thermocycler with the heated lid set at ≥ 75 °C as follows.
00:30:00 at 20 °C
00:30:00 at 65 °C
Hold at 4 °C
Proceed immediately to Adaptor Ligation.
Adaptor Ligation
Adaptor Ligation
[ ! ] Dilute the red NEBNext Adaptor* prior to setting up the ligation reaction in ice-cold Adaptor Dilution Buffer and keep the 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 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 24.
| Ligation Reaction | Volume Per One Library | |
| End Prepped DNA (Step 24) | 60 µl | |
| Diluted Adaptor (Step 25) | 2.5 µl | |
| NEBNext Ligation Enhancer | 1 µl | |
| 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.
NEBNext Ligation EnhancerNew England BiolabsCatalog #E7374
NEBNext Ultra II Ligation Master MixNew England BiolabsCatalog #E7648
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 00:15:00 at 20 °C in a thermocycler.
Add 3 µL (blue) USER™ Enzyme to the ligation mixture from Step 60, resulting in total volume of 96.5 µL
NEBNext USER EnzymeNew England BiolabsCatalog #E7458
Mix well and incubate at 37 °C for 00:15:00 with the heated lid set to ≥ 45 °C
Proceed immediately to Purification of the Ligation Reaction.
Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads
Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads
[ ! ] Note: If you are selecting for libraries with larger insert size (> 200 nt) follow the size selection recommendations in Appendix A, Chapter 6.
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.
NEBNext Sample Purification BeadsNew England BiolabsCatalog #E7767
Incubate for 00:10:00 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 (~00:05:00 ), discard the supernatant that contains unwanted fragments.
Caution: Do not discard beads.
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.
80% Ethanol (freshly prepared)
Repeat Step 35 once for a total of 2 washing steps.
go to step #35 Repeat Step
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 17 µL 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 00:02:00 at room temperature. Put the tube in the magnet until the solution is clear.
Without disturbing the bead pellet, transfer 15 µL of the supernatant to a clean PCR tube and proceed to PCR enrichment.
Note
If you need to stop at this point in the protocol, samples can be stored at –20°C.
PCR Enrichment of Adaptor Ligated DNA
PCR Enrichment of Adaptor Ligated DNA
[ ! ] 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:
| Component | Volume Per One Library | |
| Adaptor Ligated DNA (Step 40) | 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:
| Component | Volume Per One Library | |
| Adaptor ligated DNA (Step 40) | 15 µl | |
| NEBNext Ultra II Q5 Master Mix | 25 µl | |
| Index (X)/i7 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 44.A and Table 44.B):
Table 44.A:
| Cycle Step | Temp | Time | Cycles | |
| Initial Denaturation | 98°C | 30 seconds | 1 | |
| Denaturation | 98°C | 10 seconds | 6–13*,** | |
| Annealing/Extension | 65°C | 75 seconds | ||
| Final Extension | 65°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 44.B: Recommended PCR cycles based on total RNA input amount:
| Total RNA Input | Recommended PCR Cycles | |
| 1,000 ng | 6–7 | |
| 50 ng | 7–8 | |
| 10 ng | 9–10 | |
| 1 ng | 12–13 |
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.
Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads
Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads
Vortex SPRIselect Beads or NEBNext Sample Purification Beads to resuspend.
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.
NEBNext Sample Purification BeadsNew England BiolabsCatalog #E6315
Incubate for 00:05:00 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 (~00:05:00 ), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Caution: Do not discard beads.
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.
80% Ethanol (freshly prepared)
Repeat Step 49 once for a total of 2 washing steps.
go to step #49 Repeat Step
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 23 µL 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 00:02:00 at room temperature. Place the tube in the magnetic rack until the solution is clear.
Transfer20 µL of the supernatant to a clean PCR tube, and store at –20°C.
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 128 bp (adaptor-dimer) is visible in the bioanalyzer traces, bring up the sample volume (from Step 53) to 50 μl with 0.1X TE buffer and repeat the SPRIselect Bead or NEBNext Sample Purification Bead Cleanup Step (Section "Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads").
Figure 56: Example of RNA library size distribution on a Bioanalyzer.