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Home > ÀüÁ¦Ç°º¸±â > NGS °ü·Ã > Immune-Seq > Mouse BCR IgG H/K/L Profiling Kit(v1)
Mouse B-cell receptorÀÇ Heavy chain, Light chain ¼­¿­ ºÐ¼®

Mouse BCR IgG H/K/L Profiling Kit(v1)

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Clontech
634422
SMARTer¢ç Mouse BCR IgG H/K/L Profiling Kit
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12ȸ
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Clontech
634423
SMARTer¢ç Mouse BCR IgG H/K/L Profiling Kit
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48 ȸ
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6,356,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â
Clontech
634424
SMARTer¢ç Mouse BCR IgG H/K/L Profiling Kit
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96 ȸ
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10,162,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â

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º» Á¦Ç°Àº SMART-Seq¢ç Mouse BCR (with UMIs) (Code 634351-634353) Á¦Ç°À¸·Î ¾÷±×·¹ÀÌµå µÇ¾ú½À´Ï´Ù.
ÀÚ¼¼ÇÑ ³»¿ëÀº ´ÙÄ«¶óÄÚ¸®¾Æ (02-2081-2510)·Î ¹®ÀÇ ÁÖ¼¼¿ä.

  • Mouse B cell receptor (BCR)ÀÇ ±¸¼º¿øÀÎ IgGÀÇ Heavy chain (H), Light chain (Kappa chain (¥ê), Lamda chain (¥ë))¿¡ ´ëÇÑ NGS ¼­¿­ ºÐ¼®
  • ÇϳªÀÇ »ùÇÿ¡¼­ Heavy chain (H), Kappa chain (¥ê), Lamda chain (¥ë)À» Á¤¹ÐÇÏ°Ô °ËÃâ ¹× ºÐ¼®
  • Input sample: Spleen, lymph node, PBMCs, hybridoma¿¡¼­ ÃßÃâÇÑ 10ng - 3§¶ total RNA
  • ³ôÀº ƯÀ̼º°ú °¨µµ·Î BCR profiling
Á¦Ç°¼³¸í
B cellÀº ÀûÀÀ¸é¿ª¹ÝÀÀ (adaptive immune response)¿¡¼­ ÇʼöÀûÀÎ ¿ªÇÒÀ» ¼öÇàÇϸç, B cell Ç¥¸é¿¡´Â º´¿øüÀÇ Æ¯ÀÌÀûÀÎ ºÐÀÚ ÆÐÅÏÀ» ÀÎÁöÇÏ´Â B cell receptors (BCRs)¸¦ ¹ßÇöÇÏ°í ÀÖ´Ù. BCR repertoire profiling (ƯÀÌÀûÀÎ H, K, L gene segments¸¦ ¹ßÇöÇÏ´Â receptor¿Í clonotypeÀÇ ´Ù¾ç¼ºÀ» °ËÃâ)Àº °Ç°­ÇÑ °³Ã¼ÀÇ ÀûÀÀ¸é¿ª½Ã½ºÅÛ¿¡ ´ëÇÑ °íÂû»Ó¸¸ ¾Æ´Ï¶ó, ¿©·¯ Áúȯ°ú ¿¬°üÀÌ µÇ¾î ÀÖ´Ù. ±×·¯¹Ç·Î ¸é¿ª½Ã½ºÅÛ¿¡ ÀÇÇÏ¿© ¹ßÇöµÇ´Â clonotype°ú isotypeÀ» Á¤È®ÇÏ°Ô µ¿Á¤ÇÔÀ¸·Î½á B cell repertoire¸¦ ºÐ¼®ÇÒ ¼ö ÀÖ´Ù.
SMARTer¢ç Mouse BCR IgG H/K/L Profiling Kit´Â 5¡¯ RACE (Rapid Amplification cDNA Ends) ¹æ¹ý°ú NGS ±â¼úÀ» Á¶ÇÕÇÑ Á¦Ç°À¸·Î½á ³ôÀº ƯÀ̼º°ú °¨µµ·Î BCR profilingÀÌ °¡´ÉÇÏ´Ù. ÇϳªÀÇ ½ÇÇè ¹ÝÀÀ¿¡¼­ ´Ù¼öÀÇ PCR À¯ÀüÀÚ¸¦ ÁõÆøÇÏ´Â ±âÁ¸ÀÇ Multiplex PCRÀ» ÀÌ¿ëÇÑ ¹æ¹ýÀº ƯÁ¤ ¼­¿­À» ÁõÆøÇÔ¿¡ ÀÖ¾î °¨µµ, ƯÀ̼º, ÆíÇ⼺(bias) ¹®Á¦ µîÀÌ ¹ß»ýÇÏ´Â °ÍÀ¸·Î ¾Ë·ÁÁ® ÀÖÀ¸¸ç ÀÌ´Â isotype µ¿Á¤À» º¸´Ù ±î´Ù·Ó°Ô ¸¸µç´Ù. ¹Ý¸é, 5¡¯ RACE ¹æ½ÄÀº BCR heavy chain ¶Ç´Â light chainÀÇ constant region ¿µ¿ªÀ» priming ÇÏ¿© variable regionÀ» ÁõÆøÇϹǷÎ, º¸´Ù ÆíÇ⼺ÀÌ ÀûÀº BCR profilingÀÌ °¡´ÉÇÏ´Ù.


±×¸² 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.


±×¸² 2. SMARTer Mouse BCR IgG H/K/L Profiling Kit workflow.
Panel A. dT-primed first-strand cDNA synthesis is followed by two rounds of successive PCR for amplification of cDNA sequences. After post-PCR purification, size selection, and quality analysis, the library is ready for sequencing.
Panel B. Two rounds of successive PCR for the amplification of cDNA sequences corresponding to variable regions of BCR IgG heavy chain or BCR light chain (kappa or lamda) transcripts.


±×¸² 3. 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.


±×¸² 4. 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.
Applications
Mouse BCR repertoire analysis (heavy and light [kappa and lambda] chains)
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  • Package 1: -70 ¡É
    Package 2: -20 ¡É
  • 10X Lysis Buffer, Nuclease-Free Water:-20¡É º¸Á¸. À¶ÇØ ÈÄ, 4 ¡É º¸Á¸
    Elution Buffer: -20 ¡É º¸Á¸. À¶ÇØ ÈÄ ½Ç¿Â º¸Á¸
  • Mouse BCR IgG H/K/L Indexing Primer Set HT for Illumina: -20 ¡É
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  • SMARTer Mouse BCR IgG H/K/L Profiling Kit Components
  • Mouse BCR IgG H/K/L Indexing Primer Set HT for Illumina -12
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Keyword : BCR,B cell receptor,repertoire,variable region,Immune cell,Immune-seq,Immunology,,SMART,smart-seq,NGS,Next generation sequencing,cancer,Cancer research,¾Ï¿¬±¸,vaccine,¹é½Å,¹é½Å¿¬±¸

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