TITLE: NEBNext® Immune Sequencing Kit (Human) (E6320)
AUTHORS: maria.matos  , New  England   Biolabs

[DESCRIPTION]
This protocol details NEBNext® Immune Sequencing Kit (Human).

https://www.neb.com/-/media/nebus/files/manuals/manuale6320.pdf?rev=1dca310ed12a4e89b322063d31db030d&hash=D4D7E9FBED23CC7448447137DBFF8C0B

[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.

[STEPS]
SECTION: NEBNext Immune Sequencing Reverse Transcription and cDNA Synthesis
1. 
 
Mix the following components in a sterile nuclease-free tube:


 

SECTION: NEBNext Immune Sequencing Reverse Transcription and cDNA Synthesis
2. 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. 


 


SECTION: NEBNext Immune Sequencing Reverse Transcription and cDNA Synthesis
3. Place in a thermal cycler, with the heated lid set to ≥ 80 °C, and run the following program:


 

SECTION: Purify the cDNA with Streptavidin Magnetic Beads
4. Prepare Bind and Wash Buffer (1X) to be used in Step 14.
SECTION: Purify the cDNA with Streptavidin Magnetic Beads
4.1. Dilute NEBNext IS Bind and Wash Buffer (2X) to 1X with an equal volume of nuclease-free water (not provided).
 

SECTION: Purify the cDNA with Streptavidin Magnetic Beads
5. 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.

SECTION: Purify the cDNA with Streptavidin Magnetic Beads
6. Place the tube on a magnetic rack at -20 Room temperature. Once the solution is clear (~2 min) carefully remove and discard the supernatant without disturbing the bead pellet.

SECTION: Purify the cDNA with Streptavidin Magnetic Beads
7. Remove the tube from the magnet and wash the beads by adding 200 µLof 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.

SECTION: Purify the cDNA with Streptavidin Magnetic Beads
8. Place the tube on a magnetic rack. Once the solution is clear, carefully remove and discard the supernatant without disturbing the bead pellet.
SECTION: Purify the cDNA with Streptavidin Magnetic Beads
9. Remove the tube from the magnet and wash the beads by adding 200 µLof 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.
SECTION: Purify the cDNA with Streptavidin Magnetic Beads
10. 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.

SECTION: Purify the cDNA with Streptavidin Magnetic Beads
11. Add 20 µL beads to the cDNA products (20.5 uL) from Step 3 Vortex briefly to mix.

SECTION: Purify the cDNA with Streptavidin Magnetic Beads
12. Place the tube on a rotator for 15 min at 80 Room temperature.

SECTION: Purify the cDNA with Streptavidin Magnetic Beads
13. 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.
SECTION: Purify the cDNA with Streptavidin Magnetic Beads
14. 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.


 



SECTION: Purify the cDNA with Streptavidin Magnetic Beads
15. 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.
SECTION: Purify the cDNA with Streptavidin Magnetic Beads
16. Remove the tube from the magnetic rack and add 100 µL of 0.1% Tween. Vortex briefly to resuspend the beads.


 


SECTION: Purify the cDNA with Streptavidin Magnetic Beads
17. 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. 

SECTION: Purify the cDNA with Streptavidin Magnetic Beads
18. 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.

SECTION: Purify the cDNA with Streptavidin Magnetic Beads
19. Place the tube in a thermal cycler, with the heated lid set at 105 °C, and run the following program:

 

SECTION: Purify the cDNA with Streptavidin Magnetic Beads
20. Remove the tube from the thermal cycler, vortex briefly, spin down the contents and place on the magnetic rack. 
SECTION: Purify the cDNA with Streptavidin Magnetic Beads
21.  Once the solution is clear, transfer 21 µL of the supernatant into a new tube and discard the beads.


 





SECTION: PCR1 for VDJ Region Amplification and Index Barcode Incorporation
22. 
 

SECTION: PCR1 for VDJ Region Amplification and Index Barcode Incorporation
23.  Add the following components to a sterile strip tube:
        COMPONENT  VOLUME    Purified cDNA (Step 2.18)  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  
 
SECTION: PCR1 for VDJ Region Amplification and Index Barcode Incorporation
23.  Add the following components to a sterile strip tube:
        COMPONENT  VOLUME    Purified cDNA (Step 2.18)  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 TCR Primers (Human)  2 µl    (blue) NEBNext i7 Primer*  1 µl    (white) Nuclease-free Water  14 µl    Total Volume  50 µl  
 
SECTION: PCR1 for VDJ Region Amplification and Index Barcode Incorporation
23.  
        COMPONENT  VOLUME    Purified cDNA (Step 2.18)  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 IS TCR Primers (Human)  2 µl    (blue) NEBNext i7 Index Primer*  1 µl    (white) Nuclease-free Water  12 µl    Total Volume  50 µl  
 
SECTION: PCR1 for VDJ Region Amplification and Index Barcode Incorporation
24. 
SECTION: PCR1 for VDJ Region Amplification and Index Barcode Incorporation
25. 
SECTION: PCR1 for VDJ Region Amplification and Index Barcode Incorporation
23.  Add the following components to a sterile strip tube:
        COMPONENT  VOLUME    Purified cDNA (Step 2.18)  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 IS TCR Primers (Human)  2 µl    (blue) NEBNext i7 Index Primer*  1 µl    (white) Nuclease-free Water  12 µl    Total Volume  50 µl  
 
SECTION: PCR1 for VDJ Region Amplification and Index Barcode Incorporation
24. 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.
SECTION: PCR1 for VDJ Region Amplification and Index Barcode Incorporation
25. Place the tube on a thermal cycler and perform PCR amplification using the following PCR cycling program:
 
            CYCLE STEP  TEMP  TIME  CYCLES    Initial Denaturation  98°C  60 seconds  1    Denaturation  98°C  10 seconds      Annealing  64°C  30 seconds  12    Extension  72°C  30 seconds      Final Extension  72°C  60 seconds  1    Hold  4°C  ∞    


SECTION: Cleanup of PCR1 Amplification
26. 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 105 Room temperature for at least 30 min before use.
SECTION: Cleanup of PCR1 Amplification
27. Add 50 µL of resuspended NEBNext Sample Purification Beads to each 50 µl PCR reactions. Mix well by pipetting or brief vortexing.

SECTION: Cleanup of PCR1 Amplification
28. Incubate for 5 min at Room temperature.

SECTION: Cleanup of PCR1 Amplification
29. Quickly spin the tube in a microcentrifuge and place it on a magnetic rack. Once the solution is clear (~5 min) carefully remove and discard the supernatant without disturbing the bead pellet.
 


SECTION: Cleanup of PCR1 Amplification
30. Wash beads by adding 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.

SECTION: Cleanup of PCR1 Amplification
31. Wash beads by adding 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 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.
SECTION: Cleanup of PCR1 Amplification
32. Air dry the beads with more emphasis for up to 5 min while the tube is on the magnetic stand with the lid open.
 

  

SECTION: Cleanup of PCR1 Amplification
33. 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 2 min at -20 Room temperature. Quickly spin the tube and place it on a magnetic rack to separate beads from supernatant.

SECTION: Cleanup of PCR1 Amplification
34. After the solution is clear (about 2 minutes), carefully transfer 23 µL supernatant to a new PCR tube.
 


SECTION: qPCR for PCR2 Cycle Optimization
35. 
 
Add the following components to a sterile strip tube: 
        COMPONENT  VOLUME    Purified PCR1 DNA (Step 4.9)  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  
 
SECTION: qPCR for PCR2 Cycle Optimization
36. 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.
SECTION: qPCR for PCR2 Cycle Optimization
37. Place the tube on a qPCR instrument and run the following PCR cycling program: 
            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*)  30    Hold  4°C  ∞    
 

SECTION: qPCR for PCR2 Cycle Optimization
38. 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).

  

SECTION: PCR2 for Final Library
39. Add the following components to a sterile strip tube: 
        COMPONENT  VOLUME    Purified PCR1 DNA (Step 4.9)  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  
 
SECTION: PCR2 for Final Library
40. 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.
SECTION: PCR2 for Final Library
41. Place the tube on a thermal cycler and run the following PCR cycling program:
 
            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  ∞    
 
Table 1. For enriched B Cell or T Cell RNA input, PCR2 cycle number is recommended as in the table:
 
          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  
 
 


SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
42. 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 -20 Room temperature for at least 30 min before use.

SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
43. 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.

SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
44. Incubate for 5 min at -20 Room temperature.

SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
45. 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.

SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
46. Add 15 µL of resuspended NEBNext Sample Purification Beads to supernatant. Mix well by pipetting or brief vortexing.

SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
47. Incubate for 5 min at -20 Room temperature.

SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
48. Quickly spin the tube in a microcentrifuge and place it on a magnetic rack. Once the solution is clear (~5 min) carefully remove and discard the supernatant without disturbing the bead pellet. Do not discard beads.

SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
49. Wash beads by adding 200 µL of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at -20 Room temperature for 30 s, and then carefully remove and discard the supernatant.

SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
50. Wash beads by adding 200 µL of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at -20 Room temperature for 30 s, 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.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
51. Air dry the beads for up to 5 min while the tube is on the magnetic stand with the lid open.

 

  

SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
52. 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 2 min at -20 Room temperature.

SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
53. 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.
 

SECTION: Assess Library Quality on a Bioanalyzer High Sensitivity Chip or TapeStation High Sensitivity D1000 ScreenTape®
54. Dilute library 5-fold in nuclease-free water.
SECTION: Assess Library Quality on a Bioanalyzer High Sensitivity Chip or TapeStation High Sensitivity D1000 ScreenTape®
55. Run 1 µL on a Bioanalyzer DNA High Sensitivity chip.

SECTION: Assess Library Quality on a Bioanalyzer High Sensitivity Chip or TapeStation High Sensitivity D1000 ScreenTape®
56. 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).
  
  
  

SECTION: Sequencing and Data Analysis
57. 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.

 

SECTION: Index Pooling Guidelines
58. For more detailed indexing information please refer to the manual for NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1), NEB #E7600.
https://www.neb.com/-/media/nebus/files/manuals/manuale7600.pdf?rev=66702217f5614fae834024da17e8009b&hash=10C7820E5CB9543B5446A574B2DF2B31

For multiplexing samples, use Table 1 for some valid index combinations.

   
  
  

SECTION: PCR1 for VDJ Region Amplification and Index Barcode Incorporation
24. 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.
SECTION: PCR1 for VDJ Region Amplification and Index Barcode Incorporation
25. Place the tube on a thermal cycler and perform PCR amplification using the following PCR cycling program:
 
        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  ∞    
 
SECTION: Cleanup of PCR1 Amplification
26. 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 30 min before use.

SECTION: Cleanup of PCR1 Amplification
27. Add 50 µL of resuspended NEBNext Sample Purification Beads to each 50 µl PCR reactions. Mix well by pipetting or brief vortexing.

SECTION: Cleanup of PCR1 Amplification
28. Incubate for 5 min at Room temperature.
SECTION: Cleanup of PCR1 Amplification
29. Quickly spin the tube in a microcentrifuge and place it on a magnetic rack. Once the solution is clear (~5 min) carefully remove and discard the supernatant without disturbing the bead pellet.
 

SECTION: Cleanup of PCR1 Amplification
30. Wash beads by adding 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.
SECTION: Cleanup of PCR1 Amplification
31. Wash beads by adding 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 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.
SECTION: Cleanup of PCR1 Amplification
32. Air dry the beads with more emphasis for up to 5 min while the tube is on the magnetic stand with the lid open.
 
  

SECTION: Cleanup of PCR1 Amplification
33. 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 2 min at -20 Room temperature. Quickly spin the tube and place it on a magnetic rack to separate beads from supernatant.
SECTION: Cleanup of PCR1 Amplification
34. After the solution is clear (about 2 minutes), carefully transfer 23 µL supernatant to a new PCR tube.
 

SECTION: qPCR for PCR2 Cycle Optimization
35. 
 
Add the following components to a sterile strip tube:  
        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  
 
SECTION: qPCR for PCR2 Cycle Optimization
36. 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.
SECTION: qPCR for PCR2 Cycle Optimization
37. Place the tube on a qPCR instrument and run the following PCR cycling program: 
        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  ∞    
 
SECTION: qPCR for PCR2 Cycle Optimization
38. 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).
  

SECTION: PCR2 for Final Library
39. Add the following components to a sterile strip tube: 
        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  
 
SECTION: PCR2 for Final Library
40. 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.
SECTION: PCR2 for Final Library
41. Place the tube on a thermal cycler and run the following PCR cycling program: 
        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  ∞    
 Table 1. For enriched B Cell or T Cell RNA input, PCR2 cycle number is recommended as in the table: 
        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  
 
 

SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
42. 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 -20 Room temperature for at least 30 min before use.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
43. 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.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
44. Incubate for 5 min at -20 Room temperature.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
45. 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.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
46. Add 15 µL of resuspended NEBNext Sample Purification Beads to supernatant. Mix well by pipetting or brief vortexing.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
47. Incubate for 5 min at -20 Room temperature.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
48. Quickly spin the tube in a microcentrifuge and place it on a magnetic rack. Once the solution is clear (~5 min) carefully remove and discard the supernatant without disturbing the bead pellet. Do not discard beads.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
49. Wash beads by adding 200 µL of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at -20 Room temperature for 30 s, and then carefully remove and discard the supernatant.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
50. Wash beads by adding 200 µL of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at -20 Room temperature for 30 s, 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.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
51. Air dry the beads for up to 5 min while the tube is on the magnetic stand with the lid open.
 
  

SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
52. 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 2 min at -20 Room temperature.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
53. 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.
 

SECTION: Assess Library Quality on a Bioanalyzer High Sensitivity Chip or TapeStation High Sensitivity D1000 ScreenTape®
54. Dilute library 5-fold in nuclease-free water.
SECTION: Assess Library Quality on a Bioanalyzer High Sensitivity Chip or TapeStation High Sensitivity D1000 ScreenTape®
55. Run 1 µL on a Bioanalyzer DNA High Sensitivity chip.
SECTION: Assess Library Quality on a Bioanalyzer High Sensitivity Chip or TapeStation High Sensitivity D1000 ScreenTape®
56. 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).
  
  
  

SECTION: Sequencing and Data Analysis
57. 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.
 

SECTION: Index Pooling Guidelines
58. For more detailed indexing information please refer to the manual for NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1), NEB #E7600.
https://www.neb.com/-/media/nebus/files/manuals/manuale7600.pdf?rev=66702217f5614fae834024da17e8009b&hash=10C7820E5CB9543B5446A574B2DF2B31

For multiplexing samples, use Table 1 for some valid index combinations.

  
  
  

SECTION: PCR1 for VDJ Region Amplification and Index Barcode Incorporation
24. 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.
SECTION: PCR1 for VDJ Region Amplification and Index Barcode Incorporation
25. Place the tube on a thermal cycler and perform PCR amplification using the following PCR cycling program: 
      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  ∞    
 
SECTION: Cleanup of PCR1 Amplification
26. 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 105 Room temperature for at least 30 min before use.
SECTION: Cleanup of PCR1 Amplification
27. Add 50 µL of resuspended NEBNext Sample Purification Beads to each 50 µl PCR reactions. Mix well by pipetting or brief vortexing.
SECTION: Cleanup of PCR1 Amplification
28. Incubate for 5 min at Room temperature.
SECTION: Cleanup of PCR1 Amplification
29. Quickly spin the tube in a microcentrifuge and place it on a magnetic rack. Once the solution is clear (~5 min) carefully remove and discard the supernatant without disturbing the bead pellet.
 

SECTION: Cleanup of PCR1 Amplification
30. Wash beads by adding 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.
SECTION: Cleanup of PCR1 Amplification
31. Wash beads by adding 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 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.
SECTION: Cleanup of PCR1 Amplification
32. Air dry the beads with more emphasis for up to 5 min while the tube is on the magnetic stand with the lid open.
 
  

SECTION: Cleanup of PCR1 Amplification
33. 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 2 min at -20 Room temperature. Quickly spin the tube and place it on a magnetic rack to separate beads from supernatant.
SECTION: Cleanup of PCR1 Amplification
34. After the solution is clear (about 2 minutes), carefully transfer 23 µL supernatant to a new PCR tube.
 

SECTION: qPCR for PCR2 Cycle Optimization
35. 
 
Add the following components to a sterile strip tube: 
        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  
 
SECTION: qPCR for PCR2 Cycle Optimization
36. 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.
SECTION: qPCR for PCR2 Cycle Optimization
37. Place the tube on a qPCR instrument and run the following PCR cycling program: 
        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  ∞    
 
SECTION: qPCR for PCR2 Cycle Optimization
38. 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).
  

SECTION: PCR2 for Final Library
39. Add the following components to a sterile strip tube: 
        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  
 
SECTION: PCR2 for Final Library
40. 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.
SECTION: PCR2 for Final Library
41. Place the tube on a thermal cycler and run the following PCR cycling program:
 
        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  ∞    
 Table 1. For enriched B Cell or T Cell RNA input, PCR2 cycle number is recommended as in the table: 
        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  
 
 

SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
42. 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 -20 Room temperature for at least 30 min before use.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
43. 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.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
44. Incubate for 5 min at -20 Room temperature.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
45. 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.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
46. Add 15 µL of resuspended NEBNext Sample Purification Beads to supernatant. Mix well by pipetting or brief vortexing.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
47. Incubate for 5 min at -20 Room temperature.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
48. Quickly spin the tube in a microcentrifuge and place it on a magnetic rack. Once the solution is clear (~5 min) carefully remove and discard the supernatant without disturbing the bead pellet. Do not discard beads.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
49. Wash beads by adding 200 µL of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at -20 Room temperature for 30 s, and then carefully remove and discard the supernatant.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
50. Wash beads by adding 200 µL of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at -20 Room temperature for 30 s, 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.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
51. Air dry the beads for up to 5 min while the tube is on the magnetic stand with the lid open.
 
  

SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
52. 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 2 min at -20 Room temperature.
SECTION: Cleanup of PCR2 Amplification to Obtain Final Libraries
53. 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.
 

SECTION: Assess Library Quality on a Bioanalyzer High Sensitivity Chip or TapeStation High Sensitivity D1000 ScreenTape®
54. Dilute library 5-fold in nuclease-free water.
SECTION: Assess Library Quality on a Bioanalyzer High Sensitivity Chip or TapeStation High Sensitivity D1000 ScreenTape®
55. Run 1 µL on a Bioanalyzer DNA High Sensitivity chip.
SECTION: Assess Library Quality on a Bioanalyzer High Sensitivity Chip or TapeStation High Sensitivity D1000 ScreenTape®
56. 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).
  
  
  

SECTION: Sequencing and Data Analysis
57. 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.

 

SECTION: Index Pooling Guidelines
58. For more detailed indexing information please refer to the manual for NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1), NEB #E7600.
https://www.neb.com/-/media/nebus/files/manuals/manuale7600.pdf?rev=66702217f5614fae834024da17e8009b&hash=10C7820E5CB9543B5446A574B2DF2B31

For multiplexing samples, use Table 1 for some valid index combinations.