Abstract

This work was supported by Wellcome Trust Grant 096388, JDRF Grant 9-2011-253, the National Institute for Health Research Cambridge Biomedical Research Centre (BRC) and Award P01AI039671 (to LSW. and JAT.) from the National Institute of Allergy and Infectious Diseases (NIAID). CW is supported by the Wellcome Trust (089989). The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of NIAID or the National Institutes of Health. The Cambridge Institute for Medical Research is in receipt of Wellcome Trust Strategic Award 100140. We gratefully acknowledge the participation of all NIHR Cambridge BioResource volunteers. We thank the Cambridge BioResource staff for their help with volunteer recruitment. We thank members of the Cambridge BioResource SAB and Management Committee for their support of our study and the National Institute for Health Research Cambridge Biomedical Research Centre for funding. We thank Fay Rodger and Ruth Littleboy for running the Illumina MiSeq in the Molecular Genetics Laboratories, Addenbrooke's Hospital, Cambridge. This research was supported by the Cambridge NIHR BRC Cell Phenotyping Hub. In particular, we wish to thank Anna Petrunkina Harrison, Simon McCallum, Christopher Bowman, Natalia Savinykh, Esther Perez and Jelena Markovic Djuric for their advice and support in cell sorting. We also thank Helen Stevens, Pamela Clarke, Gillian Coleman, Sarah Dawson, Jennifer Denesha, Simon Duley, Meeta Maisuria-Armer and Trupti Mistry for acquisition and preparation of samples.

Highlights

  • A subset of regulatory CD4+ T cells (Tregs) is characterized by the stable constitutive expression of the transcription factor FOXP3

  • We developed a nextgeneration sequencing (NGS) method to assess at single-base resolution the methylation status of the FOXP3 Tregspecific demethylated region (TSDR)

  • The method, which we originally developed to genotype rs1800521 single nucleotide polymorphism in the gene AIRE (Methods detailed as Supporting Information), reports at single base resolution the methylation of each DNA amplicon, provides hundreds to thousands of reads per replicate and can be multiplexed to analyze several DNA regions simultaneously

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Summary

Introduction

A subset of regulatory CD4+ T cells (Tregs) is characterized by the stable constitutive expression of the transcription factor FOXP3. Several studies have used a PCR cloning and sequencing method to assess the methylation of each CpG site within the TSDR, this is a laborious method and normally less than 50 clones per sample are analyzed [3, 9, 10].

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