SMARTer Mouse BCR IgG H/K/L Profiling Kit—capturing complete V(D)J variable regions of BCR transcripts

The SMARTer Mouse BCR IgG H/K/L Profiling Kit pairs 5' RACE with NGS technology to provide a sensitive, accurate, and optimized approach to BCR profiling. The 5'-RACE method reduces variability and allows for priming from the constant region of BCR heavy or light chains. This kit combines these benefits with gene-specific amplification to capture complete V(D)J variable regions of BCR transcripts and provide a highly sensitive and reproducible method for profiling B-cell repertoires.

The kit is designed to work with a range of RNA input amounts depending on the sample type, and has been shown to generate high-quality, Illumina-ready libraries from as little as 10 ng to 3 µg of total RNA from spleen, lymph node, peripheral blood mononuclear cells (PBMCs), and hybridomas.

Downstream sequencing of H/K/L chains allows for accurate identification of top clonotypes and reliable assignment of isotype in a majority of cases.

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Cat. #	Product	Size	Price
634422	SMARTer® Mouse BCR IgG H/K/L Profiling Kit	12 Rxns	$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 Development of B-cell receptors 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. 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 Specific amplification of IgG subclasses and kappa and lambda light chains Accurate amplification of all five subclasses of IgG. Libraries containing BCR heavy and light chain (kappa) sequences were generated using the SMARTer Mouse BCR IgG H/K/L Profiling kit, starting with 10 ng of RNA isolated from an in-house hybridoma (10E8) and two ATCC hybridoma samples (HB-8117 and TIB-127) with different IgG subtypes. Panel A. Bioanalyzer traces show gene-specific amplification of heavy or light chains of each hybridoma. In each sample, the expected peak between 700–900 bp is observed. The strong discrete peaks in the electropherograms show that the kappa chain is being expressed in these hybridomas. In contrast, the lambda chain sequencing library is comparatively broad and of a smaller size range than expected. Peaks labeled "LM" and "UM" correspond to DNA reference markers included in each analysis. Panel B. Mapping metrics determined using MiXCR software (version 1.8), aligned against all IG sequences. Output from the MiXCR software for the top two clones shows the V, D, and J alleles and the isotype and subclass as identified by the software. Back Optimized pooling strategy for library amplification PCR cycling and pooling workflow. For each sample, after RT of all mRNAs in the sample, the user may specifically amplify 1–3 chains of IgG. Each amplification uses 5 µl of the RT. Following the first PCR, 1 µl of each PCR is used in a separate PCR 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 the Bioanalyzer or Fragment Analyzer. The user may then choose which amplified chain to pool for sequencing. Back Flexibility in PCR pooling for different experimental requirements PCR pooling affects alignment and on-target percentages. Libraries containing BCR heavy and light chain sequences were generated using the SMART Mouse BCR kit as described in the user manual. If only heavy and kappa light chain PCR products are pooled and sequenced ("HK" in the table), a high percentage of the sequences align to IG reference sequences (using MiXCR software version 1.8) for each sample. If the lambda light chain PCR products are also pooled ("HKL" in the table), alignment percentages drop. However, the majority heavy chain and kappa chain sequences (top two clones) are identical and in the same proportion for each sequencing sample. CDR3 amino acid sequence, which defines the affinity of the antibody, is given for each clonotype shown.
634423	SMARTer® Mouse BCR IgG H/K/L Profiling Kit	48 Rxns	$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 Development of B-cell receptors 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. 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 Specific amplification of IgG subclasses and kappa and lambda light chains Accurate amplification of all five subclasses of IgG. Libraries containing BCR heavy and light chain (kappa) sequences were generated using the SMARTer Mouse BCR IgG H/K/L Profiling kit, starting with 10 ng of RNA isolated from an in-house hybridoma (10E8) and two ATCC hybridoma samples (HB-8117 and TIB-127) with different IgG subtypes. Panel A. Bioanalyzer traces show gene-specific amplification of heavy or light chains of each hybridoma. In each sample, the expected peak between 700–900 bp is observed. The strong discrete peaks in the electropherograms show that the kappa chain is being expressed in these hybridomas. In contrast, the lambda chain sequencing library is comparatively broad and of a smaller size range than expected. Peaks labeled "LM" and "UM" correspond to DNA reference markers included in each analysis. Panel B. Mapping metrics determined using MiXCR software (version 1.8), aligned against all IG sequences. Output from the MiXCR software for the top two clones shows the V, D, and J alleles and the isotype and subclass as identified by the software. Back Optimized pooling strategy for library amplification PCR cycling and pooling workflow. For each sample, after RT of all mRNAs in the sample, the user may specifically amplify 1–3 chains of IgG. Each amplification uses 5 µl of the RT. Following the first PCR, 1 µl of each PCR is used in a separate PCR 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 the Bioanalyzer or Fragment Analyzer. The user may then choose which amplified chain to pool for sequencing. Back Flexibility in PCR pooling for different experimental requirements PCR pooling affects alignment and on-target percentages. Libraries containing BCR heavy and light chain sequences were generated using the SMART Mouse BCR kit as described in the user manual. If only heavy and kappa light chain PCR products are pooled and sequenced ("HK" in the table), a high percentage of the sequences align to IG reference sequences (using MiXCR software version 1.8) for each sample. If the lambda light chain PCR products are also pooled ("HKL" in the table), alignment percentages drop. However, the majority heavy chain and kappa chain sequences (top two clones) are identical and in the same proportion for each sequencing sample. CDR3 amino acid sequence, which defines the affinity of the antibody, is given for each clonotype shown.
634424	SMARTer® Mouse BCR IgG H/K/L Profiling Kit	96 Rxns	$4915.00
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 Development of B-cell receptors 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. 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 Specific amplification of IgG subclasses and kappa and lambda light chains Accurate amplification of all five subclasses of IgG. Libraries containing BCR heavy and light chain (kappa) sequences were generated using the SMARTer Mouse BCR IgG H/K/L Profiling kit, starting with 10 ng of RNA isolated from an in-house hybridoma (10E8) and two ATCC hybridoma samples (HB-8117 and TIB-127) with different IgG subtypes. Panel A. Bioanalyzer traces show gene-specific amplification of heavy or light chains of each hybridoma. In each sample, the expected peak between 700–900 bp is observed. The strong discrete peaks in the electropherograms show that the kappa chain is being expressed in these hybridomas. In contrast, the lambda chain sequencing library is comparatively broad and of a smaller size range than expected. Peaks labeled "LM" and "UM" correspond to DNA reference markers included in each analysis. Panel B. Mapping metrics determined using MiXCR software (version 1.8), aligned against all IG sequences. Output from the MiXCR software for the top two clones shows the V, D, and J alleles and the isotype and subclass as identified by the software. Back Optimized pooling strategy for library amplification PCR cycling and pooling workflow. For each sample, after RT of all mRNAs in the sample, the user may specifically amplify 1–3 chains of IgG. Each amplification uses 5 µl of the RT. Following the first PCR, 1 µl of each PCR is used in a separate PCR 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 the Bioanalyzer or Fragment Analyzer. The user may then choose which amplified chain to pool for sequencing. Back Flexibility in PCR pooling for different experimental requirements PCR pooling affects alignment and on-target percentages. Libraries containing BCR heavy and light chain sequences were generated using the SMART Mouse BCR kit as described in the user manual. If only heavy and kappa light chain PCR products are pooled and sequenced ("HK" in the table), a high percentage of the sequences align to IG reference sequences (using MiXCR software version 1.8) for each sample. If the lambda light chain PCR products are also pooled ("HKL" in the table), alignment percentages drop. However, the majority heavy chain and kappa chain sequences (top two clones) are identical and in the same proportion for each sequencing sample. CDR3 amino acid sequence, which defines the affinity of the antibody, is given for each clonotype shown.

Overview

Input: 10 ng–3 µg of total RNA from spleen, lymph node, PBMCs, and hybridomas
Sensitive and specific clonotype detection with optimized cDNA library generation
Accurate amplification of mouse IgG subclasses and identification via sequencing in a majority of cases
Optimized PCR pooling strategy for highly sensitive detection of different chains from the same sample
Flexible pooling strategies accommodate different experimental requirements regarding alignment and identification of primary chain sequences

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Applications

Mouse BCR repertoire analysis (heavy and light [kappa and lambda] chains)

Additional product information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.

Specific, accurate amplification for BCR repertoire profiling

The SMARTer Mouse BCR IgG H/K/L Profiling Kit leverages SMART technology and employs a 5' RACE-like approach to capture complete V(D)J variable regions of BCR transcripts. Read the tech note for key technical features and workflow advantages, as well as application data on amplification of heavy and light chains, clonotype detection, and PCR pooling strategies.

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