Mar 24, 2022
NEBNext® Immune Sequencing Kit (Human) (E6320)
- 1New England Biolabs
Protocol Citation: New England Biolabs 2022. NEBNext® Immune Sequencing Kit (Human) (E6320). protocols.io https://dx.doi.org/10.17504/protocols.io.q26g74b83gwz/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: January 04, 2022
Last Modified: March 24, 2022
Protocol Integer ID: 56531
Keywords: NEBNext® Immune Sequencing Kit , Human
Abstract
This protocol details NEBNext® Immune Sequencing Kit (Human).
Guidelines
Overview
Immune repertoire sequencing is a powerful tool that can be used to analyze the immune response to diseases and pathogens from current and past exposures. Areas of particular interest include characterization of autoimmune diseases, oncology, discovery of neutralizing antibodies against infectious disease, or use as a diagnostic tool for residual disease detection.
In order to create the incredible genetic diversity required for antigen recognition by B and T Cells, lymphocytes construct unique antigen receptors via a process known as V(D)J recombination. In this process, each cell selects a single V, D, and J gene segment through genetic recombination, introducing additional non-germline-encoded nucleotides at the junctions. This process generates immune receptor diversity, the majority of which is encoded in the heavy chain complementarity determining region 3 (CDR3) (Figure 1).
Recent improvements in the read length and throughput of next-generation sequencing (NGS) platforms have resulted in a rise in the popularity of immune repertoire sequencing. The complex structure and sequence diversity of antibody-encoding genes have provided significant challenges to the development of a simple and reliable method to sequence immune receptor sequences.
The NEBNext Immune Sequencing Library Preparation Kit (Figure 2) provides all the components required for enrichment and sequencing of the B Cell Receptor (BCR) and T Cell Receptor (TCR) RNA transcripts present in a sample. NEBNext Immune Sequencing Kit has been developed and optimized to provide accurate sequencing of full-length immune transcript repertoires of B-cells and T-cells. This allows for exhaustive somatic mutation profiling across complete V, D and J segments, full isotype information analysis (IgM, IgD, IgG, IgA and IgE), and also allows for synthesis and expression of complete antibody chains for downstream immunological assays. In addition, this approach allows characterization of TRA and TRB chains. The method uses unique molecular identifier scheme specifically designed to barcode each mRNA molecule, allowing PCR copies derived from an individual mRNA to be collapsed into a single consensus sequence. This improves sequence accuracy by resolving PCR and sequencing errors. This also eliminates PCR bias, thus allowing for quantitative digital molecule counting.
Defining features of the Immune Sequencing method include:
a) Full-length variable sequences are generated (including isotype information), allowing downstream antibody synthesis and functional characterization not possible with approaches sequencing only the CDR3 region.
b) Variable region primers are not required, which reduces primer pool complexity and allows for the unbiased and simultaneous recovery of B Cell and T Cell receptor transcripts.
c) Unique molecular barcoding approach minimizes PCR biases and improves sequencing accuracy by allowing a consensus to be generated from duplicate sequencing reads originating from the same transcript. Moreover, unique barcoding enables accurate quantitation of each clone present in the sample.
d) High target capture efficiency allows for immune repertoire sequencing and analysis from sub-microgram quantities of total RNA.
Step I-Prepare target RNA
Immune Sequencing does not require mRNA isolation prior to library construction. Total RNA can be prepared from any sample or tissue known to contain B Cells or T Cells, such as peripheral blood mononuclear cells, bone marrow, lymph nodes, or any other tissue in which infiltrating lymphocytes are present. Cells or tissues can be frozen, but intact, high quality RNA is required for optimal performance.
Step II-Perform molecular-tagging reverse transcription
During this 50-minute reaction full-length mRNA is reverse transcribed and the 3´ end of each cDNA is tagged with a DNA adaptor containing a unique molecular identifier (UMI) barcode.
Step III-Purify with Streptavidin Magnetic Beads
This 30-minute cleanup step removes all salts, reagents and unused cDNA adaptor oligonucleotides (TSO and RT Primers) allowing clean downstream amplification steps.
Step IV-Primary amplification of target fragments
This primary PCR step uses one primer which binds the universal cDNA adaptor and a primer mix targeting constant regions of the desired immune receptors. Products are then purified with Sample Purification Beads.
Step V-Secondary PCR cycle optimization with real-time PCR
This optional real-time PCR allows accurate assessment of target quantity leading to an optimized choice of PCR cycles for the final library PCR.
Step VI-Secondary PCR including Illumina® adaptor addition
This secondary PCR adds additional Illumina adaptor sequences to the target fragments (including Illumina multiplexing index sequences if desired). After Sample Purification Bead cleanup the libraries are ready for Bioanalyzer or TapeStation analysis, quantification and sequencing on the MiSeq platform.
Applications
The NEBNext Immune Sequencing Kit (Human) contains enzymes and buffers that are ideal to convert a small amount of human RNA input into indexed libraries for next-generation sequencing on the Illumina® MiSeq platform (Illumina, Inc). The workflow of NEBNext Immune Sequencing (Human) Kit is very user-friendly and fast with minimal hands-on time. Each of these components must pass rigorous quality control standards and are lot controlled, both individually and as a set of reagents.
For larger volume requirements, customized and bulk packaging is available by purchasing through the OEM/Bulks department at NEB. Please contact OEM@neb.com for further information.
Materials
The Kit Includes:
NEB #E6320S contains E2628S (Pouch with BCR and TCR primers), #E6325-1 (Box 1 of 2), and #E6325-2 (Box 2 of 2) and is sufficient for preparation of up to 24 reactions. NEB #E6320L contains E2628L (Pouch with BCR and TCR primers), #E6325-3 (Box 1 of 2), and #E6325-4 (Box 2 of 2) and is sufficient for preparation of up to 96 reactions. All reagents should be stored at t –20°C, 4°C or Room temperature as indicated below. Colored bullets represent the color of the cap of the tube containing the reagent.
NEB #E2628S/L, NEBNext Immune Sequencing Primers (Human)
Pouch: Store at -20°C.
- (blue) NEBNext IS BCR Primers (Human)
- (blue) NEBNext IS TCR Primers (Human)
NEB #E6325-1,
NEBNext Immune Sequencing Kit RT and PCR Reagents (24 reactions)New England BiolabsCatalog #E6320S
Box 1 of 2: Store at -20°C.
- (lilac) NEBNext IS RT Buffer (4X)
- (lilac) NEBNext IS RT Primer
- (lilac) NEBNext IS TS Oligos
- (lilac) NEBNext First Strand Synthesis Enzyme Mix
- (lilac) dNTP Solution Mix
- (lilac) NEBNext Cell Lysis Buffer
- (blue) Q5® Reaction Buffer (5X)
- (blue) Q5 Hot Start High-Fidelity DNA Polymerase
- (blue) NEBNext i701-i706 Primers
- (blue) NEBNext i501-i504 Primers
- (blue) NEBNext IS PCR2 Universal Primer
- (white) Nuclease-free Water
- (white) TE (0.1X)
NEB #E6325-3
NEBNext® Immune Sequencing Kit (Human) 96 reactionsNew England BiolabsCatalog #E6320L
Box 1 of 2: Store at -20°C.
- lilac) NEBNext IS RT Buffer (4X)
- (lilac) NEBNext IS RT Primer
- (lilac) NEBNext IS TS Oligos
- (lilac) NEBNext First Strand Synthesis Enzyme Mix
- (lilac) dNTP Solution Mix
- (lilac) NEBNext Cell Lysis Buffer
- (blue) Q5 Reaction Buffer (5X)
- (blue) Q5 Hot Start High-Fidelity DNA Polymerase
- (blue) NEBNext i701-i712 Primers
- (blue) NEBNext i501-i508 Primers
- (blue) NEBNext IS PCR2 Universal Primer
- (white) Nuclease-free Water
- (white) TE (0.1X)
NEB #E6325-2/#E6325-4, NEBNext Immune Sequencing Kit cDNA Purification Reagents
Box 2 of 2: Store at 4°C. Do not freeze.
- (white) Streptavidin Magnetic Beads
- (white) NEBNext IS Bind and Wash Buffer (2X)
- (white) 0.1% Tween
- (white) NEBNext Sample Purification Beads*
*This component should be stored at Room temperature.
Required Materials Not Included
SYBR® GreenThermo Fisher ScientificCatalog #S7563
• 80% Ethanol (freshly prepared)
DNA LoBind TubesEppendorfCatalog ##022431021
NEBNext® Magnetic Separation RackNew England BiolabsCatalog #S1515S or
96S Super MagnetAlpaquaCatalog #A001322
• Thermal cycler
• qPCR machine and associated 96 well plates
• Bioanalyzer®/TapeStation® and associated consumables
• MiSeq® and v3 600 cycle sequencing reagent
• Nuclease-free Water
• Tube rotating mixer
6-Tube Magnetic Separation RackNew England BiolabsCatalog #S1506S
• DNase RNase-free PCR strip tubes (USA Scientific 1402-1708)
Kit Components
NEB #E6320S Table of Components
| A | B | C | |
| NEB # | PRODUCT | VOLUME | |
| E6321A | NEBNext IS RT Buffer (4X) | 0.120 ml | |
| E6322A | NEBNext Cell Lysis Buffer | 0.012 ml | |
| E6323A | dNTP Solution Mix | 0.108 ml | |
| E6324A | NEBNext IS RT Primer | 0.024 ml | |
| E6326A | NEBNext IS TS Oligos | 0.024 ml | |
| E7761A | NEBNext First Strand Synthesis Enzyme Mix | 0.048 ml | |
| E6327A | Hot Start High-Fidelity DNA Polymerase | 0.060 ml | |
| E6328A | Q5 Reaction Buffer (5X) | 0.600 ml | |
| E2626A | NEBNext IS BCR Primers (Human) | 0.048 ml | |
| E2627A | NEBNext IS TCR Primers (Human) | 0.048 ml | |
| E6329A | NEBNext IS PCR2 Universal Primer | 0.072 ml | |
| E6355A | Streptavidin Magnetic Beads | 0.360 ml | |
| E6356A | NEBNext IS Bind and Wash Buffer (2X) | 11.3 ml | |
| E6357A | 0.1% Tween | 3.0 ml | |
| E6358A | NEBNext Sample Purification Beads | 2.8 ml | |
| E6331A | TE (0.1X) | 2.3 ml | |
| E6332A | Nuclease-free Water | 1.5 ml | |
| E6333A | NEBNext i701 Primer | 0.008 ml | |
| E6334A | NEBNext i702 Primer | 0.008 ml | |
| E6335A | NEBNext i703 Primer | 0.008 ml | |
| E6336A | NEBNext i704 Primer | 0.008 ml | |
| E6337A | NEBNext i705 Primer | 0.008 ml | |
| E6338A | NEBNext i706 Primer | 0.008 ml | |
| E6339A | NEBNext i501 Primer | 0.036 ml | |
| E6340A | NEBNext i502 Primer | 0.036 ml | |
| E6341A | NEBNext i503 Primer | 0.036 ml | |
| E6342A | NEBNext i504 Primer | 0.036 ml |
NEB #E6320L Table of Components
| A | B | C | |
| NEB # | PRODUCT | VOLUME | |
| E6321AA | NEBNext IS RT Buffer (4X) | 0.480 ml | |
| E6322AA | NEBNext Cell Lysis Buffer | 0.048 ml | |
| E6323AA | dNTP Solution Mix | 0.432 ml | |
| E6324AA | NEBNext IS RT Primer | 0.096 ml | |
| E6326AA | NEBNext IS TS Oligos | 0.096 ml | |
| E7761AA | NEBNext First Strand Synthesis Enzyme Mix | 0.192 ml | |
| E6327AA | Q5 Hot Start High-Fidelity DNA Polymerase | 0.240 ml | |
| E6328AA | Q5 Reaction Buffer (5X) | 2.400 ml | |
| E2626AA | NEBNext IS BCR Primers (Human) | 0.192 ml | |
| E2627AA | NEBNext IS TCR Primers (Human) | 0.192 ml | |
| E6329AA | NEBNext IS PCR2 Universal Primer | 0.288 ml | |
| E6355AA | Streptavidin Magnetic Beads | 1.440 ml | |
| E6356AA | NEBNext IS Bind and Wash Buffer (2X) | 45.2 ml | |
| E6357AA | 0.1% Tween | 11.8 ml | |
| E6358AA | NEBNext Sample Purification Beads | 11.1 ml | |
| E6331AA | TE (0.1X) | 9.2 ml | |
| E6332AA | Nuclease-free Water | 5.8 ml | |
| E6333AA | NEBNext i701 Primer | 0.032 ml | |
| E6334AA | NEBNext i702 Primer | 0.032 ml | |
| E6335AA | NEBNext i703 Primer | 0.032 ml | |
| E6336AA | NEBNext i704 Primer | 0.032 ml | |
| E6337AA | NEBNext i705 Primer | 0.032 ml | |
| E6338AA | NEBNext i706 Primer | 0.032 ml | |
| E6347AA | NEBNext i707 Primer | 0.032 ml | |
| E6348AA | NEBNext i708 Primer | 0.032 ml | |
| E6349AA | NEBNext i709Primer | 0.032 ml | |
| E6352AA | NEBNext i710 Primer | 0.032 ml | |
| E6353AA | NEBNext i711 Primer | 0.032 ml | |
| E6354AA | NEBNext i712 Primer | 0.032 ml | |
| E6339AA | NEBNext i501 Primer | 0.144 ml | |
| E6340AA | NEBNext i502 Primer | 0.144 ml | |
| E6341AA | NEBNext i503 Primer | 0.144 ml | |
| E6342AA | NEBNext i504 Primer | 0.144 ml | |
| E6343AA | NEBNext i505 Primer | 0.144 ml | |
| E6344AA | NEBNext i506 Primer | 0.144 ml | |
| E6345AA | NEBNext i507 Primer | 0.144 ml | |
| E6346AA | NEBNext i508 Primer | 0.144 ml |
NEBNext Immune Sequencing Reverse Transcription and cDNA Synthesis
NEBNext Immune Sequencing Reverse Transcription and cDNA Synthesis
Note
RNA Sample Recommendations: The RNA sample should be free of salts (e.g., Mg2+, or guanidine salts) or organics (e.g., phenol and ethanol). High quality RNA with a RIN > 7 is recommended for optimal performance.
Starting Material: 10 ng –1 µg enriched B cell or T Cell or B cell and T Cell total RNA, or 10 ng –1 µg total RNA from peripheral blood mononuclear cells (PBMCs). The maximum volume of RNA input is 9 µL .
Mix the following components in a sterile nuclease-free tube:
| A | B | |
| COMPONENT | VOLUME | |
| (lilac) NEBNext IS RT Buffer (4X) | 5 µl | |
| (lilac) NEBNext IS RT Primer | 1 µl | |
| (lilac) NEBNext IS TS Oligos | 1 µl | |
| (lilac) dNTP Solution Mix | 2 µl | |
| (lilac) NEBNext First Strand Synthesis Enzyme Mix | 2 µl | |
| (lilac) NEBNext Cell Lysis Buffer | 0.5 µl | |
| Total RNA | 1-9 µl | |
| (white) Nuclease-free Water | Variable | |
| Total Volume | 20.5 µl |
Set a 100 µl or 20 µl pipette to 15 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 the performance.
Place in a thermal cycler, with the heated lid set to ≥ 80 °C , and run the following program:
| A | B | C | |
| CYCLE STEP | TEMP | TIME | |
| Incubation | 42°C | 40 minutes | |
| Inactivation | 70°C | 10 minutes | |
| Hold | 4°C | ∞ |
Purify the cDNA with Streptavidin Magnetic Beads
Purify the cDNA with Streptavidin Magnetic Beads
Prepare Bind and Wash Buffer (1X) to be used in Step 14.
Dilute NEBNext IS Bind and Wash Buffer (2X) to 1X with an equal volume of nuclease-free water (not provided).
Note
For each sample, you will need 100 µL Bind and Wash Buffer (1X).
Vortex Streptavidin Magnetic Beads to resuspend. Aliquot the total amount of Streptavidin Magnetic Beads needed (15 µL of Streptavidin Magnetic Beads per sample) into a clean RNase-free 1.5 mL tube.
Place the tube on a magnetic rack at Room temperature . Once the solution is clear (~00:02:00 ) carefully remove and discard the supernatant without disturbing the bead pellet.
2m
Remove the tube from the magnet and wash the beads by adding 200 µL of undiluted NEBNext IS Bind and Wash Buffer (2X). Vortex briefly to resuspend the beads and quickly spin the tube in a microcentrifuge to collect any sample on the sides of the tube.
Place the tube on a magnetic rack. Once the solution is clear, carefully remove and discard the supernatant without disturbing the bead pellet.
Remove the tube from the magnet and wash the beads by adding 200 µL of undiluted NEBNext IS Bind and Wash Buffer (2X). Vortex briefly to resuspend the beads and quickly spin the tube in a microcentrifuge to collect any sample on the sides of the tube. Place the tube on a magnetic rack. Once the solution is clear, carefully remove and discard the supernatant without disturbing the bead pellet for a total of two washes.
Remove the tube from the magnetic rack. Resuspend the beads in 20 µL of undiluted NEBNext IS Bind and Wash Buffer (2X) for each sample (e.g., for 4 samples, add 4 x 20 = 80 µL NEBNext IS Bind and Wash Buffer (2X). Vortex briefly to resuspend the beads.
Add 20 µL beads to the cDNA products (20.5 uL) from Step 3 Vortex briefly to mix.
Place the tube on a rotator for 00:15:00 at Room temperature .
15m
Quickly spin down the tube to collect any sample on the sides of the tube and place the tube on a magnetic rack. Once the solution is clear, carefully remove and discard the supernatant without disturbing the bead pellet.
Remove the tube from the magnetic rack. Add 100 µL diluted Bind and Wash Buffer (1X) (from Step 4). Vortex briefly to resuspend the beads.
Note
Vortexing is better than flicking the tube, as it minimizes foaming.
Quickly spin down the tube to collect any sample on the sides and place the tube on a magnetic rack. Once the solution is clear, carefully remove and discard the supernatant without disturbing the bead pellet.
Remove the tube from the magnetic rack and add 100 µL of 0.1% Tween. Vortex briefly to resuspend the beads.
Note
This step is critical to remove all the salt from the beads before elution.
Quickly spin down the tube to collect any sample on the sides of the tube and place the tubes on a magnetic rack. Once the solution is clear, carefully remove and discard the supernatant without disturbing the bead pellet. With a 20 ul pipette tip, carefully remove any residual liquid left at the bottom of the tube without disturbing the beads.
Remove the tube from the magnet and add 23 µL of 0.1% Tween. Vortex briefly to resuspend the beads and quickly spin in a microcentrifuge to collect any sample on the sides of the tube.
Place the tube in a thermal cycler, with the heated lid set at 105 °C , and run the following program:
| A | B | C | |
| CYCLE STEP | TEMP | TIME | |
| Incubation | 95°C | 3 minutes | |
| Hold | 25°C | ∞ |
Remove the tube from the thermal cycler, vortex briefly, spin down the contents and place on the magnetic rack.
Once the solution is clear, transfer 21 µL of the supernatant into a new tube and discard the beads.
Note
Safe Stopping Point:
It is safe to store the cDNA at -20 °C Overnight .
Note
- Use Option 22.1 for enriching B Cell Receptor (BCR) chains.
- Use Option 22.2 for enriching T Cell Receptor (TCR) chains.
- Use Option 22.3 for enriching both BCR chains and TCR chains.
B Cell Receptor Chains PCR 136 steps
Add the following components to a sterile strip tube:
| A | B | |
| COMPONENT | VOLUME | |
| Purified cDNA (Step 21) | 21 µl | |
| (blue) Q5 Reaction Buffer (5X) | 10 µl | |
| (lilac) dNTP Solution Mix | 1 µl | |
| (blue) Q5 Hot Start High-Fidelity DNA Polymerase | 1 µl | |
| (blue) NEBNext IS BCR Primers (Human) | 2 µl | |
| (blue) NEBNext i7 Index Primer* | 1 µl | |
| (white) Nuclease-free Water | 14 µl | |
| Total Volume | 50 µl |
*Use only one i7 Primer per sample
Set a 100 ul or 200 ul pipette to 40 ul 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.
Place the tube on a thermal cycler and perform PCR amplification using the following PCR cycling program:
| A | B | C | D | |
| CYCLE STEP | TEMP | TIME | CYCLES | |
| Initial Denaturation | 98°C | 60 seconds | 1 | |
| Denaturation | 98°C | 10 seconds | 12 | |
| Annealing | 64°C | 30 seconds | ||
| Extension | 72°C | 30 seconds | ||
| Final Extension | 72°C | 60 seconds | 1 | |
| Hold | 4°C | ∞ |
Cleanup of PCR1 Amplification
Cleanup of PCR1 Amplification
41m
41m
Vortex NEBNext Sample Purification Beads to resuspend. SPRIselect and AMPure® XP Beads can be used as well. If using AMPure XP Beads, please allow the beads to warm to Room temperature for at least 00:30:00 before use.
30m
Add 50 µL of resuspended NEBNext Sample Purification Beads to each 50 µl PCR reactions. Mix well by pipetting or brief vortexing.
Incubate for 00:05:00 at Room temperature .
5m
Quickly spin the tube in a microcentrifuge and place it on a magnetic rack. Once the solution is clear (~00:05:00 ) carefully remove and discard the supernatant without disturbing the bead pellet.
Note
Caution: Do not discard beads.
5m
Wash beads by adding 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.
30s
Wash beads by adding 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 once for a total of two wash steps. After supernatant is discarded, use a 20 µl pipette tip to carefully remove any residual liquid left at the bottom of the tube without disturbing the beads.
30s
Air dry the beads with more emphasis for up to 00:05:00 while the tube is on the magnetic stand with the lid open.
Note
Caution: Do not over dry the beads. This may result in lower recovery of DNA target.
5m
Elute DNA target from beads by adding 25 µL TE (0.1X) to beads. Mix well on a vortex mixer or by pipetting up and down at least 10 times. Incubate for at least 00:02:00 at Room temperature . Quickly spin the tube and place it on a magnetic rack to separate beads from supernatant.
2m
After the solution is clear (about 2 minutes), carefully transfer 23 µL supernatant to a new PCR tube.
Note
Safe Stopping Point: It is safe to store the PCR product overnight at -20 °C .
qPCR for PCR2 Cycle Optimization
qPCR for PCR2 Cycle Optimization
Note
The qPCR step is optional; it is only used to identify the optimal final PCR cycling conditions, as each sample can contain varying amounts of amplifiable molecules. One can achieve a similar result by manually taking an aliquot of the PCR reaction every 5 cycles and visualizing each aliquot on Bioanalyzer or TapeStation to identify the optimal cycling number that yields the highest product with the lowest background. The number of cycles should be high enough to provide sufficient library fragments for a successful sequencing run, but low enough to avoid PCR artifacts and over-cycling (high molecular weight fragments on Bioanalyzer). Typically, most samples will amplify best in the range of 6–18 cycles.
Add the following components to a sterile strip tube:
| A | B | |
| COMPONENT | VOLUME | |
| Purified PCR1 DNA (Step 34) | 5 µl | |
| (blue) Q5 Reaction Buffer (5X) | 5 µl | |
| (lilac) dNTP Solution Mix | 0.5 µl | |
| (blue) Q5 Hot Start High-Fidelity DNA Polymerase | 0.5 µl | |
| (blue) NEBNext IS PCR2 Universal Primer | 1 µl | |
| (blue) NEBNext i5 Primer* | 1 µl | |
| SYBR Green (20X)** | 0.25 µl | |
| (white) Nuclease-free Water | 11.75 µl | |
| Total Volume | 25 µl |
* Use one i5 Primer per PCR reaction
** 20X working stock of SYBR Green can be made by diluting 1 µl (10,000X) SYBR Green (Life Technologies #S7563) into 399 µl DMSO and stored at 4°C for up to a month. You only need 0.2 x SYBR final in the qPCR reaction.
Set a 100 µl or 20 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.
Place the tube on a qPCR instrument and run the following PCR cycling program:
| A | B | C | D | |
| CYCLE STEP | TEMP | TIME | CYCLES | |
| Initial Denaturation | 98°C | 60 seconds | 1 | |
| Denaturation | 98°C | 10 seconds | 30 | |
| Annealing and Extension | 72°C | 30 seconds (fluorescence on*) | ||
| Hold | 4°C | ∞ |
* Select the SYBR channel.
The amplification curve for each sample is used to determine the optimal number of PCR cycles to use during PCR2 (Section: PCR2 for Final Library). The PCR cycle number is approximated by determining the cycle number (round down to the nearest whole number) corresponding to 2/3 of the peak fluorescence (see example in Figure 3; in this example 13 PCR cycles will be used in Section: PCR2 for Final Library).
PCR2 for Final Library
PCR2 for Final Library
Add the following components to a sterile strip tube:
| A | B | |
| COMPONENT | VOLUME | |
| Purified PCR1 DNA (Step 34) | 10 µl | |
| (blue) Q5 Reaction Buffer (5X) | 10 µl | |
| (lilac) dNTP Solution Mix | 1 µl | |
| (blue) Q5 Hot Start High-Fidelity DNA Polymerase | 1 µl | |
| (blue) NEBNext IS PCR2 Universal Primer | 2 µl | |
| (blue) NEBNext i5 Index Primer* | 2 µl | |
| (white) Nuclease-free Water | 24 µl | |
| Total Volume | 50 µl |
* Use one i5 Primer per PCR reaction
Set a 100 µl pipette to 40 µl 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.
Place the tube on a thermal cycler and run the following PCR cycling program:
| A | B | C | D | |
| CYCLE STEP | TEMP | TIME | CYCLES | |
| Initial Denaturation | 98°C | 60 seconds | 1 | |
| Denaturation | 98°C | 10 seconds | X* | |
| Annealing and Extension | 72°C | 30 seconds | ||
| Hold | 4°C | ∞ |
* Use number of cycles determined from Step 38. If qPCR was not done, see Table 1 for recommended number of PCR cycles. In this case the number of PCR cycles should be chosen based on input amount and sample type. The number of cycles should be high enough to provide sufficient library fragments for a successful sequencing run, but low enough to avoid PCR artifacts and over-cycling (high molecular weight fragments on Bioanalyzer).
Table 1. For enriched B Cell or T Cell RNA input, PCR2 cycle number is recommended as in the table:
| A | B | C | |
| TOTAL RNA INPUT | PCR1 ASSAY PCR2 | CYCLE NUMBER | |
| B Cell RNA 1,000 ng | BCR | 6-7 | |
| B Cell RNA 100 ng | BCR | 9-10 | |
| B Cell RNA 10 ng | BCR | 13-14 | |
| T Cell RNA 1,000 ng | TCR | 10-11 | |
| T Cell RNA 100 ng | TCR | 12-13 | |
| T Cell RNA 10 ng | TCR | 17-18 |
Note
If B and T cells are combined, choose a PCR2 cycle number in between the recommendations for B cell and T cell RNA.
Cleanup of PCR2 Amplification to Obtain Final Libraries
Cleanup of PCR2 Amplification to Obtain Final Libraries
46m
46m
Vortex NEBNext Sample Purification Beads to resuspend. SPRIselect and AMPure XP Beads can be used as well. If using AMPure XP Beads, please allow the beads to warm to Room temperature for at least 00:30:00 before use.
30m
Add 50 µL of TE (0.1X) to each 50 µl PCR reaction to obtain 100 µl mixture, and then add 50 µL resuspended NEBNext Sample Purification Beads to the 100 µl mixture. Mix well by pipetting or brief vortexing.
Incubate for 00:05:00 at Room temperature .
5m
Quickly spin the tube in a microcentrifuge and place it on a magnetic rack. Once the solution is clear (~5 minutes) carefully transfer the 150 µL supernatant to a new tube without disturbing the bead pellet. Save the supernatant and discard beads.
Add 15 µL of resuspended NEBNext Sample Purification Beads to supernatant. Mix well by pipetting or brief vortexing.
Incubate for 00:05:00 at Room temperature .
5m
Quickly spin the tube in a microcentrifuge and place it on a magnetic rack. Once the solution is clear (~00:05:00 ) carefully remove and discard the supernatant without disturbing the bead pellet. Do not discard beads.
5m
Wash beads by adding 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.
30s
Wash beads by adding 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 once for a total of two wash steps. After supernatant is discarded, carefully remove any residual liquid left at the bottom of the tube without disturbing the beads with a 10 µl pipette tip.
30s
Air dry the beads for up to 00:05:00 while the tube is on the magnetic stand with the lid open.
Note
Caution: Do not over dry the beads. This may result in lower recovery of DNA target.
5m
Elute DNA target from beads by adding 20 µL TE (0.1X) to beads. Mix well on a vortex mixer or by pipetting up and down at least 10 times. Incubate for at least 00:02:00 at Room temperature .
2m
Quickly spin the tube and place it on a magnetic rack to separate beads from supernatant. After the solution is clear (about 2 minutes), carefully transfer 18 µL supernatant to a new PCR tube.
Note
Safe Stopping Point: It is safe to store the library overnight at -20 °C .
Assess Library Quality on a Bioanalyzer High Sensitivity Chip or TapeStation High Sensitivity D1000 ScreenTape®
Assess Library Quality on a Bioanalyzer High Sensitivity Chip or TapeStation High Sensitivity D1000 ScreenTape®
Dilute library 5-fold in nuclease-free water.
Run 1 µL on a Bioanalyzer DNA High Sensitivity chip.
Check that the electropherogram shows the expected size distribution. B Cell Receptor chain libraries will have an average size of 647 bp (Figure 4). T Cell Receptor chain libraries will have an average size of 630 bp (Figure 5). BCR + TCR chain libraries will have an average size of 632 bp (Figure 6).
Sequencing and Data Analysis
Sequencing and Data Analysis
Sequencing should be performed on an Illumina MiSeq, using Illumina V3 sequencing chemistry. We recommend running 300 PE using a 600-cycle V3 MiSeq reagent kit with the index read between the first and second read.
Note
For information on data processing please refer to Data Usage Guideline Page within the "Usage Guidelines & Tips" panel in the "Other Tools & Resources" tab at: https://www.neb.com/products/e6320-nebnext-immune-sequencing-kit-human#Product%20Information
We have developed and optimized a pipeline for processing of data directly from FASTQ files using open-source bioinformatics tools. Details on this pipeline can be found at: https://usegalaxy.org/u/bradlanghorst/w/presto-nebnext-immune-seq-workflow-v320.
Index Pooling Guidelines
Index Pooling Guidelines
For more detailed indexing information please refer to the manual for NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1), NEB #E7600.
For multiplexing samples, use Table 1 for some valid index combinations.