Targeted Gene Bisulfite Sequencing Identifies Differential Methylation in p21

Author:

Kolli Ramya T.,Glenn Travis C.,Brown Bradley T.,Barnett Lillie M.,Lash Lawrence H.,Cummings Brian S.

Abstract

AbstractNext-generation sequencing (NGS) methods are widely available to assess methylation of whole-genomes, reduced representation of genomes, and target capture of many loci, but simple, flexible, and low-cost methods are needed to leverage NGS for sequencing single-locus amplicons from large numbers of samples. We developed a two-stage PCR approach, targeted gene bisulfite sequencing (TGBS) which uses the Illumina MiSeq and Bismark bisulfite mapper, to assess site specific changes in methylation of the cyclin-dependent kinase inhibitor p21 (CDKN1a) after exposure to a DNA methyltransferase inhibitor, 5-aza-2’-deoxycytidine (5-Aza) and determine the differences between human and rat p21 methylation. TGBS analysis of human embryonic kidney cells (HEK293) and human proximal tubular cells (hPT) demonstrated variation at a known methylation sensitive site (SIE-1), but not in rat kidney cells. Treatment of cells with 5-Aza altered the methylation of this site in correlation with increased p21 protein expression. We also found that human and rat p21 promoter sequences differ considerably in the amount of basal DNA methylation. These data showed the utility of TGBS for rapid analysis of DNA methylation of specific loci. We provide links to a ready-to-run Virtualbox that includes the program and commands for methylation analysis of bisulfite datasets, including step-by-step directions.

Publisher

Cold Spring Harbor Laboratory

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3