TCR Repertoire Analysis in Thymic Cells

Next-generation sequencing of TCR was carried out as previously described [12 (link)]. We obtained thymic CD4⁺SP cells from mice in different groups using MACS. Briefly, DNA was extracted from these thymic CD4⁺ SP cells using DNeasy Blood &Tissue kit (Qiagen), quantified using a Qubit Fluorometer (Thermo) and amplified by multiplex-PCR of rearranged variable, diverse, joining (VDJ) segments of the TCR genes, which encode the hypervariable CDR3 domain. The products were size selected using Pronex beads (Promega) and subsequently sequenced on a MiSeq (Illumina). The length and polymorphism of CDR3 were analyzed with GeneMapper 4.1 software (Thermo).

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Zhao J., Jia L., Tao Y., Zhao X., Yang J., Lu Y., Yan Y., Mao L., Hu L., Lu J., Guo M., Chen C., Zhou Y., Wen Z., He Z, & Xu L. (2023). TCR repertoire landscape reveals macrophage-mediated clone deletion in endotoxin tolerance. Inflammation Research, 72(3), 531-540.

Publication 2023

DNeasy Blood &Tissue kit Qubit Fluorometer Pronex beads GeneMapper 4.1 software

Blood Cd4 cells Mice Multiplex pcr Polymorphism Promega Tcr genes Thymic Tissue

Corresponding Organization : Soochow University

Other organizations : Guizhou Provincial Education Department

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Variable analysis

independent variables

Different groups of mice

dependent variables

Length and polymorphism of CDR3 domain in TCR sequences

control variables

Extraction of thymic CD4+ SP cells using MACS
DNA extraction using DNeasy Blood & Tissue kit
DNA quantification using Qubit Fluorometer
Amplification of rearranged VDJ segments of TCR genes by multiplex-PCR
Size selection of PCR products using Pronex beads
Sequencing on a MiSeq (Illumina) platform
Analysis of CDR3 length and polymorphism using GeneMapper 4.1 software

controls

No positive or negative controls were explicitly mentioned in the provided information.

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