![]() ![]() 100 ng of purified cDNA from each sample was sent to Adaptive Biotechnologies for the immunoSEQ hsTCRb Deep sequencing service or used for FR3AK-seq PCR. cDNA was column purified with the Oligo Clean and Concentrator Kit (Zymo Research). 4 μg each of Donor A, Donor B, and sample C RNA was reverse transcribed using a TCRB chain constant region reverse primer with Superscript III First-Strand Synthesis System (Invitrogen). ![]() 0.5 μg Donor B RNA was mixed with 4.5 μg Donor A RNA to create sample C for quantitation experiments. RNA was extracted from purified human T cells using the RNeasy Plus Minikit (Qiagen). T cells were purified from thawed PBMCs using the EasySep Human T cell enrichment kit (STEMCELL Technologies). PBMCs from Donors A and B were isolated by Ficoll-Paque (GE Healthcare) gradient centrifugation and cryopreserved. Materials and Methods 2.1 FR3 primer design We identified a cluster of related TCR sequences from patients with sporadic IBM, suggesting the presence of an antigen-driven T cell response within the muscle of these donors. ![]() Finally, we illustrate the ability of FR3AK-seq to democratize TCR repertoire sequencing by efficiently characterizing T cell responses within muscle tissue from 145 patients with IIMs and controls. We found that FR3AK-seq data, which was analyzed using open-source software, was in excellent agreement with both. We benchmark this simplified multiplex PCR-based assay, Framework Region 3 AmplifiKation Sequencing (“FR3AK-seq”), against two different industry standards for quantitative TCR repertoire sequencing: Adaptive Biotechnologies’ multiplex PCR-based hsTCRB immunoSEQ assay and ArcherDX's UMI-based Immunoverse HS TCR assay. Lower sequencing depth requirements, coupled with CDR3 analysis via single-end, short-read (100 nucleotide) sequencing, dramatically reduces the cost associated with TCR repertoire analysis. The Lancet Regional Health – Western Pacificīy designing maximally compact primer sets and a streamlined workflow, we have essentially eliminated PCR amplification bias while maintaining high sequence level accuracy.The Lancet Regional Health – Southeast Asia.The Lancet Gastroenterology & Hepatology. ![]()
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