Primer combination for simultaneously detecting T cell and B cell immune repertoire for high-throughput sequencing and kit
An immune library and primer combination technology, applied in the field of molecular biology, can solve the problems of limitations, difficulty in designing the 5' end sequence of upstream primers, poor PCR efficiency and specificity, and achieve stable efficiency, simple and convenient methods of use, and high efficiency Efficient effect
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[0113] Example 1
[0114] Using the primer combination and kit of the present invention to perform high-throughput sequencing detection, thereby simultaneously detecting T cell receptors and B cell receptors, the specific steps include the following steps:
[0115] (1) Obtain 10 mL of human blood sample in an EDTA anticoagulation tube;
[0116] (2) Use lymphocyte separation fluid Ficoll-1077 (American Sigma Company #10771) to separate peripheral blood mononuclear cells (PBMC);
[0117] (3) Using Trizol method to extract total RNA from PBMC, the reagent used is RNAzol RT (US MRC company #RN190);
[0118] (4) Use 2.0Fluorometer (U.S. Thermo Fisher Scientific company #Q32866), with RNA HS Assay Kit (Thermo Fisher Scientific, USA #Q32852) is used to determine the concentration of RNA, and then used for reverse transcription of RNA;
[0119] (5) Reverse transcription of RNA into cDNA, and add a linker at the 5'end of cDNA for the subsequent PCR amplification of the 5'end primers. The speci...
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