• Complete V(D)J sequence information - TCR mRNA의 가변영역(variable regions)의 전체 서열(Full length) 분석
• TCR-alpha, TCR-beta chain을 동시에 또는 개별적으로 NGS Library 제작 가능
• No multiplex PCR required -1회 반응당 단일 프라이머로 각각의 TCR Subunit을 증폭
• Illumina®-ready sequencing libraries - ligation-independent 방식으로 illumina® adaptor 및 index sequence 부착 가능 (Incorporate Illumina® adapter and index sequences in a ligation-independent manner, and multiplex up to 96 libraries in a single flow-cell lane)

제품설명

SMARTer® Mouse TCR a/b Profiling Kit은 T-cell receptor (TCR) repertoire의 NGS 분석을 위한 Illumina platform NGS Library 제작 키트이다. 5’-RACE 기술을 접목하여 TCR 전사체의 V(D)J 가변영역 (variable region)을 분석하며, mouse spleen, thymus, or PBMCs로부터 얻은 10ng-500ng의 total RNA 또는 1,000개-10,000개의 purified T-cell을 적용 가능하다. 본 제품을 이용하면 TCR-α, TCR-β chain을 동시에 또는 각 chain에 대한 라이브러리를 개별적으로 제작하여 분석할 수 있다.
Multiplexing PCR을 이용하는 기존의 TCR profiling 방법과 달리, library amplification의 각 반응은 single primer pair를 이용하므로 primer-dimer 생성 가능성을 낮출 수 있다. Multiplex PCR 방식은 서열특이적 편향성을 나타낼 가능성이 있으나 RACE 방식을 이용할 경우 이러한 편향성의 문제를 덜 수 있다.
본 제품은 LNA 기술이 결합된 SMART (Switching Mechanism at 5’ End of RNA Template)법을 이용하므로 민감도가 매우 높고 편향성이 적은 TCR mRNA sequence 분석을 할 수 있다.



그림 1. Library preparation workflow and PCR strategy for TCR profiling using the SMARTer approach.


그림 2. 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.


그림 3. 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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