Immunizing mice with vaccine targets and further optimization

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Library preparation workflow and PCR strategy for TCR profiling using the SMARTer approach

Library preparation workflow and PCR strategy for TCR profiling using the SMARTer approach. Panel A. Reverse transcription and PCR amplification of TCR subunit mRNA sequences. First-strand cDNA synthesis is primed by the TCR dT Primer and performed by an MMLV-derived reverse transcriptase (RT). Upon reaching the 5′ end of each mRNA molecule, the RT adds non-templated nucleotides to the first-strand cDNA. The SMART-Seq v4 Oligonucleotide contains a sequence that is complementary to the non-templated nucleotides added by the RT, and the oligo hybridizes to the first-strand cDNA. In the template-switching step, the RT uses the remainder of the SMART-Seq v4 Oligonucleotide as a template for the incorporation of an additional sequence on the end of the first-strand cDNA. Full-length variable regions of TCR cDNA are selectively amplified by PCR using primers that are complementary to the oligonucleotide-templated sequence (SMART Primer 1) and the constant region(s) of TCR-α and/or TCR-β subunits (TCRa and/or TCRb Mouse Primer 1). A subsequent round of PCR is performed to further amplify variable regions of TCR-α and/or TCR-β subunits and incorporate adapter sequences using a TCR Primer 2 Forward HT Index and a TCRa and/or TCRb Mouse Primer 2 Reverse HT Index. Included in the primers are adapter and index sequences (Read 2 + i7 + P7 and Read 1 + i5 + P5, respectively) that are compatible with the Illumina sequencing platform. Following purification, size selection, and quality analysis, the TCR cDNA library is ready for sequencing. Panel B. Semi-nested PCR approach for amplification of TCR-α and/or TCR-β subunits. The primer pairs used for the first round of amplification capture the entire variable region(s) and most of the constant region(s) of TCR-α and/or TCR-β cDNA. The second round of amplification retains the entire variable region(s) of TCR-α and/or TCR-β cDNA and a smaller portion of the constant region(s). The anticipated size of the final TCR library cDNA (inserts + adapters) is ~700–800 bp.

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Assessment of TCR diversity in mouse spleen samples with the SMARTer Mouse TCR a/b Profiling Kit

Assessment of TCR diversity in mouse spleen samples with the SMARTer Mouse TCR a/b Profiling Kit. Panel A. To compare TCR diversity between the spleen's general T-cell population and purified CD4+ T cells, total splenocytes were isolated from spleen and divided into two groups. The first group was left untouched, and the second group was subject to CD4+ T-cell purification (see Methods section for details). Total RNA was extracted from the total splenocyte group and the group enriched for CD4+ T cells, and each RNA sample was used for library preparation. Panel B. Proportion of on-target reads mapping to TCR-α or TCR-β CDR3 sequences. Panel C. Rarefaction curves showing the relationship between read depth and the number of unique clonotypes identified for TCR-α (left) and TCR-β (right). When a curve starts to flatten off, the number of unique clonotypes identified is reaching saturation. Dashed lines show the theoretical number of clonotypes as the number of reads increases.

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Assessment of TCR-α clonotype diversity in various tissues of transgenic mice of different ages with the SMARTer Mouse TCR a/b Profiling Kit

Assessment of TCR-α clonotype diversity in various tissues of transgenic mice of different ages with the SMARTer Mouse TCR a/b Profiling Kit. Panel A. The percentage of reads mapping to TCR-α CDR3 sequences identified in the thymus, spleen, peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), and large intestine (LI) is consistent across the 16-week-old, 34-week-old, and 38-week-old mice. Panel B. The immune tissues showed the highest TCR-α clonotypic diversity, while the large intestine showed the lowest diversity. This indicates that the large intestine has the lowest T-cell infiltrate. Panel C. Abundance of the TRAV3-2-TRAJ58 clonotype, expressed as a percentage of all reads mapping to any TCR-α or TCR-β clonotype, across tissue types and ages. Panel D. Abundance of the TRAV16N-TRAJ56 clonotype, expressed as a percentage of all reads mapping to any TCR-α or TCR-β clonotype, across tissue types in the 38-week-old mouse.

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634402: SMARTer Mouse TCR a/b Profiling Kit

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

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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.

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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. 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. 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. 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%.