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  • ‹ Back to Immune Profiling
  • Efficient and sensitive profiling of human B-cell receptor repertoire
  • TCRv2 kit validated for rhesus macaque samples
  • TCR repertoire profiling from mouse samples (bulk)
  • BCR repertoire profiling from mouse samples (bulk)
  • Improved TCR repertoire profiling from human samples (bulk)
  • TCR repertoire profiling from human samples (single cells)
  • BCR repertoire profiling from human samples (bulk)
SMARTer Mouse BCR IgG H/K/L Profiling Kit Visit the product page
Home › Learning centers › Next-generation sequencing › Technical notes › Immune Profiling › BCR repertoire profiling from mouse samples (bulk)

Technical notes

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    • Efficient and sensitive profiling of human B-cell receptor repertoire
    • TCRv2 kit validated for rhesus macaque samples
    • TCR repertoire profiling from mouse samples (bulk)
    • BCR repertoire profiling from mouse samples (bulk)
    • Improved TCR repertoire profiling from human samples (bulk)
    • TCR repertoire profiling from human samples (single cells)
    • BCR repertoire profiling from human samples (bulk)
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SMARTer Mouse BCR IgG H/K/L Profiling Kit Visit the product page
Tech Note

Profiling mouse B-cell receptors with SMART technology

  • 5' RACE-like approach captures complete V(D)J variable regions of BCR transcripts »
  • Flexible and optimized workflow for clonotype detection »
Introduction Results Conclusions Methods References

Introduction  

B cells are an essential part of the adaptive immune response, functioning via B-cell receptors (BCRs) expressed on their surface. Each B cell expresses a different BCR that allows it to recognize molecular patterns in pathogens. Development of BCRs (Figure 1) is a multistep process in which a progenitor cell undergoes V(D)J recombination in the germline followed by somatic hypermutation (SHM), resulting in a final product with a specific CDR3 (complementarity determining region 3) sequence in the hypervariable region of the immunoglobulin (Ig). These molecular events facilitate receptor diversity and the generation of heavy chain isotypes. Upon exposure to a stimulus or stimuli, the lambda and kappa light chain genes of the BCR undergo rearrangements to generate different light chain isotypes from the same B-cell clone.

BCR development

Figure 1. BCR development. The progenitor cell undergoes recombination of V, D, and J segments in the germline, which generates two identical heavy chains. Recombination of V and J segments generates two identical light chains. Random nucleotide additions or deletions at the junctions of the V, D, and J segments provide additional diversity. Furthermore, B cells activated by immune responses undergo somatic hypermutation (SHM), in which additional point mutations are introduced.

Understanding the profiles of BCRs, (i.e., sequencing the full-length CDR3 regions to determine the diversity of receptors and the clonotypes, defined by expression of specific heavy and light chain gene segments) can not only aid in gaining insights into the adaptive immune response in healthy individuals, but also in those with a wide range of conditions, including infectious diseases, allergies, autoimmune disorders, cancers, and aging (Yaari & Kleinstein, 2015).

Next-generation sequencing (NGS) approaches for profiling B-cell repertoires have provided valuable insights into the adaptive immune response and antibody engineering. There are two major NGS approaches used in profiling B-cell repertoires—multiplex PCR or 5' RACE. While the multiplex PCR approach allows for amplification of multiple BCR genes within one reaction, challenges with sensitivity, specificity, and biases in amplification of certain sequences can lead to difficulties in accurate identification of isotypes. On the other hand, the 5'-RACE method reduces variability and allows for priming from the constant regions of the BCR heavy or light chains. However, the burden of designing optimized primers falls onto the individual researcher.

The SMARTer Mouse BCR IgG H/K/L Profiling Kit (SMARTer mouse BCR kit) solves these problems by combining the benefits of 5' RACE with gene-specific amplification (Figure 2) to provide a highly sensitive and reproducible method for profiling B-cell repertoires. The high sensitivity of the kit accurately identifies top B-cell Ig clonotypes and reliably assigns isotype in a majority of cases, based on sequencing of the gamma (G) heavy chain and the kappa (K) and lambda (L) light chains.

SMARTer Mouse BCR IgG H/K/L Profiling Kit workflow

Figure 2. SMARTer Mouse BCR IgG H/K/L Profiling Kit workflow. Panel A. First-strand cDNA synthesis utilizes a dT-primed (BCR dT Primer) and MMLV-derived SMARTScribe Reverse Transcriptase (RT), which adds non-templated nucleotides upon reaching the 5' end of each mRNA template. The BCR oligonucleotide anneals to these non-templated nucleotides and serves as a template for the incorporation of an additional sequence of nucleotides into the first-strand cDNA by the RT. The BCR oligonucleotide contains sequence from the Illumina Read Primer 2, serves as a primer-annealing site for subsequent rounds of PCR, and ensures that only sequences from full-length cDNAs undergo amplification. Then, the library goes through a final bead clean up and the library is ready for sequencing on an Illumina MiSeq sequencer. Panel B. The first PCR uses the first-strand cDNA as a template and includes a forward primer with complementarity to the Illumina Read Primer 2 sequence (BCR Primer 1V), and a reverse primer that is complementary to the constant (i.e., non-variable) region of the BCR G heavy chain (mBCR Primer 1H), K light chain, or L light chain (mBCR Primers 1K or 1L). The chains are amplified in separate reactions. By priming from the Read Primer 2 sequence and the constant region, the first PCR specifically amplifies the entire variable region and a considerable portion of the constant region of the G K, or L chain cDNA. The second PCR takes the product from the first PCR as a template and uses semi-nested primers (mBCR Primers 2H, 2K, or 2L) to amplify the entire variable region and a portion of the constant region of BCR heavy or light chain cDNA. As in PCR 1, the BCR subunit chains are amplified in separate reactions.

The kit leverages SMART technology (Switching Mechanism at 5' End of RNA Template) and employs a 5'-RACE-like approach to capture complete V(D)J variable regions of BCR transcripts. First-strand cDNA synthesis is dT-primed, and the template-switching activity ensures that only sequences from full-length cDNAs undergo PCR amplification (Figure 2). Two rounds of PCR are then performed in succession to amplify cDNA sequences corresponding to the variable regions of the BCR chain transcripts (Figure 2, Panel B). The first PCR reaction uses first-strand cDNA as a template to amplify each BCR Ig transcript (G, K, or L) in a separate reaction. Then, the first PCR product is used as the template with semi-nested primers to amplify the entire variable region and a portion of the constant region. Following post-PCR purification, size selection, and quality analysis, the library is ready for sequencing on an Illumina MiSeq® sequencer using the 600-cycle MiSeq® Reagent Kit v3.

Results  

Specific amplification of Ig heavy (G) and light (K and L) chains

To determine if the BCR-specific primers amplified the expected regions of the BCR loci, RNA was isolated from three mouse hybridoma samples (10E8, HB-8117, and TIB-127). Then, 10 ng of RNA from each sample was used as input for the SMARTer mouse BCR kit, and the resulting libraries were analyzed for quality and sequenced. Bioanalyzer traces for each hybridoma sample show the expected peaks for the G, K, and L chains, demonstrating correct amplification (Figure 3, Panel A).

Analysis of sequencing data showed that alignment of total reads to Ig sequences were over 90% for these samples (Figure 3B). We consistently see over 75% alignment to Ig, indicating that the SMARTer mouse BCR kit produces BCR-specific libraries. The top two clones identified in our sequencing data, representing an H chain transcript and a K light chain transcript, make up >70% of the clonotype fraction combined. We also detected a small number of clones with a clone fraction of ≥0.0001. These data indicate a highly clonal BCR population, which is expected for hybridoma cell line samples. In addition, the percentage of overlapping reads was over 80%, which indicates good sequencing depth—an attribute that is potentially helpful in identifying base pair changes resulting from somatic hypermutation.

Accurate amplification of all five subclasses of IgG
B
RNA source 10E8 (Takara Bio) HB-8117 (ATCC) TIB-127 (ATCC)
Total sequencing reads 916,706 1,202,645 978,131
% successfully aligned to Ig sequences 98% 97% 93%
% overlapped 83% 83% 81%
% overlappped and aligned 82% 81% 76%
Clone fraction ≥ 0.0001 4 5 5
Clone fraction ≥ 0.0001 3 4 6
Top heavy (G) chain Distribution 50% 45% 40%
V alleles and alignment score IGHV4-1 (1252.1)
IGHV4-2
(1201.4)
IGHV9-2 (1207.3)
IGHV9-3 (1163.9)
IGHV9-1 (1078.7)
IGHV5-9 (832.6)
IGHV5-17 (815)
IGHV5-6 (799)
IGHV5-12 (764.7)
D alleles and alignment score IGHD2-4 (65) IGHD1-1 (77) IGHD5-2 (25)
IGHD5-3 (25)
IGHD5-6 (25)
J alleles and alignment score IGHJ3 (237.1) IGHJ1 (215.7) IGHJ2 (36.4)
AA seq CDR3 CARAYDYDRAWFGYW CARKSSYYGSTYVYFDVW CARHDNSGW
Top light (K) chain Distribution 46% 26% 50%
V alleles and aligment score IGKV3-4 (1342.8) IGKV3-1 (1328.2) IGKV6-29 (1352)
IGKV6-20 (1330.1)
J alleles and aligment score IGKJ5 (195.1) IGKJ1 (147.4) IGHJ2 (190.8)
AA seq CDR3 CQQSNDNPLTF CQQSRKVPSTF CGQGYSYPYTF

Figure 3. Highly specific amplification of Ig chains. Libraries containing BCR G and K chain transcripts were generated using the SMARTer Mouse BCR IgG H/K/L Profiling kit, starting with 10 ng of RNA isolated from a hybridoma developed in-house (10E8) and two manufactured ATCC hybridoma samples (HB-8117 and TIB-127). Panel A. Bioanalyzer traces showing gene-specific amplification of G, K and L chains for each hybridoma. Peaks labeled "LM" and "UM" correspond to DNA reference markers included in each analysis. Panel B. Mapping metrics were determined using MiXCR software (version 2.1.81.8; software not provided with kit) and aligned against all Ig reference sequences, with a clone fraction threshold of 0.01%. V, D, and J IMGT allele outputs, alignment scores and consensus CDR3 amino acid CDR3 for the top heavy (G) and light (K) chain clone for each hybridoma are displayed. For cases in which the MiXCR software determined the presence of more than one V, D, or J allele, all determined alleles with alignment scores are shown.

Optimized workflow for clonotype detection

The optimized workflow of the SMARTer mouse BCR kit workflow allows for flexibility in sequencing and accurate clonotype identification. Following reverse transcription, the reaction is split to allow for amplification of the G, K, and L chains in separate reactions. The second PCR adds the same sequencing indexes to each amplified Ig chain transcript for a given sample, but distinct indexes for each individual sample (e.g. G, K, L of sample 1 would all receive index A, but G, K, L of sample 2 would all receive index B). After sample validation, libraries are pooled for sequencing. This key strategy allows highly sensitive sequencing of different chains from the same sample even with a very small amount of starting material.

PCR cycling and pooling workflow

Figure 4. PCR cycling and pooling workflow. After RT step, the user amplifies the G, K, or L chain transcripts in separate reactions. Each amplification uses 5 µl of the RT reaction. Following the first PCR, 1 µl of each PCR is used in a separate PCR reaction (PCR 2) to add the same sequencing indexes to each amplified chain for a given sample, but distinct indexes for each different sample. After this final amplification in PCR2, the user may validate each product on a Bioanalyzer or other fragment analysis system. The user then has the flexibility to choose which amplified BCR chains to pool for sequencing.

We demonstrated that PCR pooling strategy affects percentage of sequencing read aligning to Ig (Figure 5). Clonal B-cell populations or hybridomas primarily express a K or L light chain —not both. The bioanalyzer traces for the K chains in our hybridoma samples contained a sharp peak representing amplification of this transcript, while the bioanalyzer traces for the L chains lacked this peak, suggesting the K light chain is the light chain expressed within our hybridoma samples (Figure 3, Panel A). Since we observed no amplification of the L chain, we omitted the L chain and pooled only the G and K chains together for sequencing (“GK” in the table below). Over 70% of sequencing reads aligned to Ig when the GK pooling strategy was used. When the L chain products were included in the pool ("GKL" in the table below), the percentage of reads aligning to Ig dropped significantly. Additionally, the proportion of the identified CDR3 sequence for the G and K chains were the same for both pooling strategies. These data demonstrate that the SMARTer mouse BCR kit allows for flexibility in sequencing approach in cases where high Ig alignment rates are required without compromising accuracy of clonotype identification.

A
% successful alignment
Hybridoma 10E8 HB-8117 TIB-127
Chains sequenced together GK GKL L GK GKL L GK GKL L
% aligned to IG 98% 37% 0.34% 98% 64% 0.46% 93% 56% 0.49%
B
Hybridoma 10E8 HB-8117 TIB-127
Sequenced chains GK GKL GK GKL GK GKL
Gamma (G) chain CDR3


%
CARAYDY
DRAWFG
YW
50%
CARAYDY
DRAWFG
YW
51%
CARKSSY
YGSTYVY
FDVW
45%
CARKSSY
YGSTYVY
FDVW
44%
CARHDNS
GW

40%
CARHDNS
GW

39%
Kappa (K) chain CDR3

%
CQQSND
NPLTF
46%
CQQSND
NPLTF
44%
CQQSRK
VPSTF
27%
CQQSRK
VPSTF
28%
CGQGYS
YPYTF
50%
CGQGYS
YPYTF
51%

Figure 5. Effect of PCR pooling strategy on Ig alignment rate and clonotype identification. G and K chain PCR products (GK) or G, K and L chain PCR products (GLK) were pooled and sequenced for each hybridoma sample. Panel A. The percentage of reads aligning to Ig reference sequences for GK pooling, GKL pooling, and L only sequencing strategies as determined by MiXCR software. Panel B. The identified CDR3 amino acid consensus sequence and percent distribution for the top heavy (G) and light (K) chain clone for GK pooling or GKL pooling strategies as determined by the MiXCR software. The clone fraction threshold was set to 0.01%.

Conclusions  

The SMARTer Mouse BCR IgG H/K/L Profiling Kit is a powerful tool for profiling mouse B-cell receptors. By leveraging SMART technology and combining a 5' RACE-like approach with gene-specific primer amplification, this workflow captures complete V(D)J variable regions of BCRs and is optimized for highly sensitive and specific clonotype detection. With primers that incorporate Illumina-specific adaptor sequences during cDNA amplification, the protocol generates indexed libraries ready for sequencing on the Illumina MiSeq platform. An additional advantage is the unique PCR cycling and pooling workflow which reduces sequencing cost while enabling accurate clonotype identification. By avoiding multiplex PCR, this kit also avoids the pitfalls of biases in amplification of certain sequences, helping to provide a complete and accurate view of mouse BCR repertoires.

Methods  

Libraries containing BCR heavy and light chain sequences were generated using the SMARTer Mouse BCR IgG H/K/L Profiling Kit as per the protocol given in the user manual. 10 ng of RNA was obtained as starting material from the indicated hybridomas (10E8, HB-8117, and TIB- 127) with different Ig subtypes. 10E8 is an in-house hybridoma; the others are ATCC lines. ATCC determined the expected isotype for their lines; 10E8 was determined in-house. Hybridomas were cultured according to established methods. Libraries were produced using the first-strand cDNA as a template in three different PCRs for the gamma heavy chain and the kappa and lambda light chains. The product of these PCRs was used as template in a set of nested PCRs, one for each chain. Following purification and size selection, libraries were validated using the Agilent 2100 Bioanalyzer. Libraries were then sequenced on an Illuminia MiSeq sequencer and analyzed using MiXCR software (version 2.1.81.8, software not provided).

References  

Bolotin, D.A. et al. MiXCR: software for comprehensive adaptive immunity profiling. Nat. Methods 12, 380–381 (2015).

Yaari, G. and Kleinstein, S.H. Practical guidelines for B-cell receptor repertoire sequencing analysis. Genome Med. 7:121 (2015).

Related Products

Cat. # Product Size License Quantity Details
634422 SMARTer® Mouse BCR IgG H/K/L Profiling Kit 12 Rxns USD $900.00

The SMARTer Mouse BCR IgG H/K/L Profiling Kit enables users to analyze B-cell receptor (BCR) diversity from total RNA samples and whole cells. This kit is designed to work with a range of RNA inputs, from 10 ng to 3 µg of total RNA obtained from 1,000 to 10,000 purified B cells. This kit can be used to generate data for both heavy (IgG only) and light chain diversity. The kit is not intended to identify the subclasses of IgG heavy chain that are expressed (i.e., IgG1, IgG2a, IgG2b, IgG2c, or IgG3).

This kit leverages SMART technology and employs a 5' RACE-like approach to capture complete V(D)J variable regions of BCR transcripts. Included in the kit are primers that incorporate Illumina-specific adaptor sequences during cDNA amplification. This kit supports up to 12 rxns.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

Back

BCR development.

BCR development.

BCR development. The progenitor cell undergoes recombination of V, D, and J segments in the germline, which generates two identical heavy chains. Recombination of V and J segments generates two identical light chains. Random nucleotide additions or deletions at the junctions of the V, D, and J segments provide additional diversity. Furthermore, B cells activated by immune responses undergo somatic hypermutation (SHM), in which additional point mutations are introduced.

Back

SMARTer Mouse BCR IgG H/K/L Profiling Kit workflow.

SMARTer Mouse BCR IgG H/K/L Profiling Kit workflow.

SMARTer Mouse BCR IgG H/K/L Profiling Kit workflow. Panel A. First-strand cDNA synthesis is dT-primed (BCR dT Primer) and performed by the MMLV-derived SMARTScribe Reverse Transcriptase (RT), which adds nontemplated nucleotides upon reaching the 5' end of each mRNA template. The BCR Oligonucleotide anneals to these nontemplated nucleotides and serves as a template for the incorporation of an additional sequence of nucleotides into the first-strand cDNA by the RT (this is the template-switching step). The BCR Oligonucleotide contains sequence from the Illumina Read Primer 2, serving as a primer-annealing site for subsequent rounds of PCR, and ensuring that only sequences from full-length cDNAs undergo amplification. Panel B. The first PCR uses the first-strand cDNA as a template and includes a forward primer with complementarity to the Illumina Read Primer 2 sequence (BCR Primer 1V), and a reverse primer that is complementary to the constant (i.e., nonvariable) region of BCR heavy or light chains (mBCR Primers 1H, 1K, or 1L). The chains are amplified in separate reactions. By priming from the Read Primer 2 sequence and the constant region, the first PCR specifically amplifies the entire variable region and a considerable portion of the constant region of BCR heavy or light chain cDNA. The second PCR takes the product from the first PCR as a template and uses semi-nested primers (mBCR Primers 2H, 2K, or 2L) to amplify the entire variable region and a portion of the constant region of BCR heavy or light chain cDNA. As in PCR 1, the BCR subunit chains are amplified in separate reactions.

Back

Highly specific amplification of Ig chains.

Highly specific amplification of Ig chains.

Highly specific amplification of Ig chains. Libraries containing BCR G and K chain transcripts were generated using the SMARTer Mouse BCR IgG H/K/L Profiling kit, starting with 10 ng of RNA isolated from a hybridoma developed in-house (10E8) and two manufactured ATCC hybridoma samples (HB-8117 and TIB-127). Panel A. Bioanalyzer traces showing gene-specific amplification of G, K and L chains for each hybridoma. Peaks labeled "LM" and "UM" correspond to DNA reference markers included in each analysis. Panel B. Mapping metrics were determined using MiXCR software (version 2.1.81.8; software not provided with kit) and aligned against all Ig reference sequences, with a clone fraction threshold of 0.01%. V, D, and J IMGT allele outputs, alignment scores and consensus CDR3 amino acid CDR3 for the top heavy (G) and light (K) chain clone for each hybridoma are displayed. For cases in which the MiXCR software determined the presence of more than one V, D, or J allele, all determined alleles with alignment scores are shown.

Back

PCR cycling and pooling workflow.

PCR cycling and pooling workflow.

PCR cycling and pooling workflow. After RT step, the user amplifies the G, K, or L chain transcripts in separate reactions. Each amplification uses 5 µl of the RT reaction. Following the first PCR, 1 µl of each PCR is used in a separate PCR reaction (PCR 2) to add the same sequencing indexes to each amplified chain for a given sample, but distinct indexes for each different sample. After this final amplification in PCR2, the user may validate each product on a Bioanalyzer or other fragment analysis system. The user then has the flexibility to choose which amplified BCR chains to pool for sequencing.

Back

Effect of PCR pooling strategy on Ig alignment rate and clonotype identification.

Effect of PCR pooling strategy on Ig alignment rate and clonotype identification.

Effect of PCR pooling strategy on Ig alignment rate and clonotype identification. G and K chain PCR products (GK) or G, K and L chain PCR products (GLK) were pooled and sequenced for each hybridoma sample. Panel A. The percentage of reads aligning to Ig reference sequences for GK pooling, GKL pooling, and L only sequencing strategies as determined by MiXCR software. Panel B. The identified CDR3 amino acid consensus sequence and percent distribution for the top heavy (G) and light (K) chain clone for GK pooling or GKL pooling strategies as determined by the MiXCR software. The clone fraction threshold was set to 0.01%.

Back

634422: SMARTer Mouse BCR IgG H/K/L Profiling Kit

634422: SMARTer Mouse BCR IgG H/K/L Profiling Kit
634423 SMARTer® Mouse BCR IgG H/K/L Profiling Kit 48 Rxns USD $3072.00

The SMARTer Mouse BCR IgG H/K/L Profiling Kit enables users to analyze B-cell receptor (BCR) diversity from total RNA samples and whole cells. This kit is designed to work with a range of RNA inputs, from 10 ng to 3 µg of total RNA obtained from 1,000 to 10,000 purified B cells. This kit can be used to generate data for both heavy (IgG only) and light chain diversity. The kit is not intended to identify the subclasses of IgG heavy chain that are expressed (i.e., IgG1, IgG2a, IgG2b, IgG2c, or IgG3).

This kit leverages SMART technology and employs a 5' RACE-like approach to capture complete V(D)J variable regions of BCR transcripts. Included in the kit are primers that incorporate Illumina-specific adaptor sequences during cDNA amplification. This kit supports up to 48 rxns.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

Back

BCR development.

BCR development.

BCR development. The progenitor cell undergoes recombination of V, D, and J segments in the germline, which generates two identical heavy chains. Recombination of V and J segments generates two identical light chains. Random nucleotide additions or deletions at the junctions of the V, D, and J segments provide additional diversity. Furthermore, B cells activated by immune responses undergo somatic hypermutation (SHM), in which additional point mutations are introduced.

Back

SMARTer Mouse BCR IgG H/K/L Profiling Kit workflow.

SMARTer Mouse BCR IgG H/K/L Profiling Kit workflow.

SMARTer Mouse BCR IgG H/K/L Profiling Kit workflow. Panel A. First-strand cDNA synthesis is dT-primed (BCR dT Primer) and performed by the MMLV-derived SMARTScribe Reverse Transcriptase (RT), which adds nontemplated nucleotides upon reaching the 5' end of each mRNA template. The BCR Oligonucleotide anneals to these nontemplated nucleotides and serves as a template for the incorporation of an additional sequence of nucleotides into the first-strand cDNA by the RT (this is the template-switching step). The BCR Oligonucleotide contains sequence from the Illumina Read Primer 2, serving as a primer-annealing site for subsequent rounds of PCR, and ensuring that only sequences from full-length cDNAs undergo amplification. Panel B. The first PCR uses the first-strand cDNA as a template and includes a forward primer with complementarity to the Illumina Read Primer 2 sequence (BCR Primer 1V), and a reverse primer that is complementary to the constant (i.e., nonvariable) region of BCR heavy or light chains (mBCR Primers 1H, 1K, or 1L). The chains are amplified in separate reactions. By priming from the Read Primer 2 sequence and the constant region, the first PCR specifically amplifies the entire variable region and a considerable portion of the constant region of BCR heavy or light chain cDNA. The second PCR takes the product from the first PCR as a template and uses semi-nested primers (mBCR Primers 2H, 2K, or 2L) to amplify the entire variable region and a portion of the constant region of BCR heavy or light chain cDNA. As in PCR 1, the BCR subunit chains are amplified in separate reactions.

Back

Highly specific amplification of Ig chains.

Highly specific amplification of Ig chains.

Highly specific amplification of Ig chains. Libraries containing BCR G and K chain transcripts were generated using the SMARTer Mouse BCR IgG H/K/L Profiling kit, starting with 10 ng of RNA isolated from a hybridoma developed in-house (10E8) and two manufactured ATCC hybridoma samples (HB-8117 and TIB-127). Panel A. Bioanalyzer traces showing gene-specific amplification of G, K and L chains for each hybridoma. Peaks labeled "LM" and "UM" correspond to DNA reference markers included in each analysis. Panel B. Mapping metrics were determined using MiXCR software (version 2.1.81.8; software not provided with kit) and aligned against all Ig reference sequences, with a clone fraction threshold of 0.01%. V, D, and J IMGT allele outputs, alignment scores and consensus CDR3 amino acid CDR3 for the top heavy (G) and light (K) chain clone for each hybridoma are displayed. For cases in which the MiXCR software determined the presence of more than one V, D, or J allele, all determined alleles with alignment scores are shown.

Back

PCR cycling and pooling workflow.

PCR cycling and pooling workflow.

PCR cycling and pooling workflow. After RT step, the user amplifies the G, K, or L chain transcripts in separate reactions. Each amplification uses 5 µl of the RT reaction. Following the first PCR, 1 µl of each PCR is used in a separate PCR reaction (PCR 2) to add the same sequencing indexes to each amplified chain for a given sample, but distinct indexes for each different sample. After this final amplification in PCR2, the user may validate each product on a Bioanalyzer or other fragment analysis system. The user then has the flexibility to choose which amplified BCR chains to pool for sequencing.

Back

Effect of PCR pooling strategy on Ig alignment rate and clonotype identification.

Effect of PCR pooling strategy on Ig alignment rate and clonotype identification.

Effect of PCR pooling strategy on Ig alignment rate and clonotype identification. G and K chain PCR products (GK) or G, K and L chain PCR products (GLK) were pooled and sequenced for each hybridoma sample. Panel A. The percentage of reads aligning to Ig reference sequences for GK pooling, GKL pooling, and L only sequencing strategies as determined by MiXCR software. Panel B. The identified CDR3 amino acid consensus sequence and percent distribution for the top heavy (G) and light (K) chain clone for GK pooling or GKL pooling strategies as determined by the MiXCR software. The clone fraction threshold was set to 0.01%.

Back

634423: SMARTer Mouse BCR IgG H/K/L Profiling Kit

634423: SMARTer Mouse BCR IgG H/K/L Profiling Kit
634424 SMARTer® Mouse BCR IgG H/K/L Profiling Kit 96 Rxns USD $5063.00

The SMARTer Mouse BCR IgG H/K/L Profiling Kit enables users to analyze B-cell receptor (BCR) diversity from total RNA samples and whole cells. This kit is designed to work with a range of RNA inputs, from 10 ng to 3 µg of total RNA obtained from 1,000 to 10,000 purified B cells. This kit can be used to generate data for both heavy (IgG only) and light chain diversity. The kit is not intended to identify the subclasses of IgG heavy chain that are expressed (i.e., IgG1, IgG2a, IgG2b, IgG2c, or IgG3).

This kit leverages SMART technology and employs a 5' RACE-like approach to capture complete V(D)J variable regions of BCR transcripts. Included in the kit are primers that incorporate Illumina®-specific adaptor sequences during cDNA amplification. This kit supports up to 96 rxns.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

Back

BCR development.

BCR development.

BCR development. The progenitor cell undergoes recombination of V, D, and J segments in the germline, which generates two identical heavy chains. Recombination of V and J segments generates two identical light chains. Random nucleotide additions or deletions at the junctions of the V, D, and J segments provide additional diversity. Furthermore, B cells activated by immune responses undergo somatic hypermutation (SHM), in which additional point mutations are introduced.

Back

SMARTer Mouse BCR IgG H/K/L Profiling Kit workflow.

SMARTer Mouse BCR IgG H/K/L Profiling Kit workflow.

SMARTer Mouse BCR IgG H/K/L Profiling Kit workflow. Panel A. First-strand cDNA synthesis is dT-primed (BCR dT Primer) and performed by the MMLV-derived SMARTScribe Reverse Transcriptase (RT), which adds nontemplated nucleotides upon reaching the 5' end of each mRNA template. The BCR Oligonucleotide anneals to these nontemplated nucleotides and serves as a template for the incorporation of an additional sequence of nucleotides into the first-strand cDNA by the RT (this is the template-switching step). The BCR Oligonucleotide contains sequence from the Illumina Read Primer 2, serving as a primer-annealing site for subsequent rounds of PCR, and ensuring that only sequences from full-length cDNAs undergo amplification. Panel B. The first PCR uses the first-strand cDNA as a template and includes a forward primer with complementarity to the Illumina Read Primer 2 sequence (BCR Primer 1V), and a reverse primer that is complementary to the constant (i.e., nonvariable) region of BCR heavy or light chains (mBCR Primers 1H, 1K, or 1L). The chains are amplified in separate reactions. By priming from the Read Primer 2 sequence and the constant region, the first PCR specifically amplifies the entire variable region and a considerable portion of the constant region of BCR heavy or light chain cDNA. The second PCR takes the product from the first PCR as a template and uses semi-nested primers (mBCR Primers 2H, 2K, or 2L) to amplify the entire variable region and a portion of the constant region of BCR heavy or light chain cDNA. As in PCR 1, the BCR subunit chains are amplified in separate reactions.

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Highly specific amplification of Ig chains.

Highly specific amplification of Ig chains.

Highly specific amplification of Ig chains. Libraries containing BCR G and K chain transcripts were generated using the SMARTer Mouse BCR IgG H/K/L Profiling kit, starting with 10 ng of RNA isolated from a hybridoma developed in-house (10E8) and two manufactured ATCC hybridoma samples (HB-8117 and TIB-127). Panel A. Bioanalyzer traces showing gene-specific amplification of G, K and L chains for each hybridoma. Peaks labeled "LM" and "UM" correspond to DNA reference markers included in each analysis. Panel B. Mapping metrics were determined using MiXCR software (version 2.1.81.8; software not provided with kit) and aligned against all Ig reference sequences, with a clone fraction threshold of 0.01%. V, D, and J IMGT allele outputs, alignment scores and consensus CDR3 amino acid CDR3 for the top heavy (G) and light (K) chain clone for each hybridoma are displayed. For cases in which the MiXCR software determined the presence of more than one V, D, or J allele, all determined alleles with alignment scores are shown.

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PCR cycling and pooling workflow.

PCR cycling and pooling workflow.

PCR cycling and pooling workflow. After RT step, the user amplifies the G, K, or L chain transcripts in separate reactions. Each amplification uses 5 µl of the RT reaction. Following the first PCR, 1 µl of each PCR is used in a separate PCR reaction (PCR 2) to add the same sequencing indexes to each amplified chain for a given sample, but distinct indexes for each different sample. After this final amplification in PCR2, the user may validate each product on a Bioanalyzer or other fragment analysis system. The user then has the flexibility to choose which amplified BCR chains to pool for sequencing.

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Effect of PCR pooling strategy on Ig alignment rate and clonotype identification.

Effect of PCR pooling strategy on Ig alignment rate and clonotype identification.

Effect of PCR pooling strategy on Ig alignment rate and clonotype identification. G and K chain PCR products (GK) or G, K and L chain PCR products (GLK) were pooled and sequenced for each hybridoma sample. Panel A. The percentage of reads aligning to Ig reference sequences for GK pooling, GKL pooling, and L only sequencing strategies as determined by MiXCR software. Panel B. The identified CDR3 amino acid consensus sequence and percent distribution for the top heavy (G) and light (K) chain clone for GK pooling or GKL pooling strategies as determined by the MiXCR software. The clone fraction threshold was set to 0.01%.

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634424: SMARTer Mouse BCR IgG H/K/L Profiling Kit

634424: SMARTer Mouse BCR IgG H/K/L Profiling Kit

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