Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Ivana Grbesa; Miriam Tannenbaum; Avital Sarusi-Portuguez; Michal Schwartz; Ofir Hakim

doi:10.3791/56313

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Method Article

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

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DOI:

10.3791/56313

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November 13th, 2017

Ivana Grbesa¹ , Miriam Tannenbaum¹ , Avital Sarusi-Portuguez¹ , Michal Schwartz¹ , Ofir Hakim¹

¹The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University

Summary

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

Abstract

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Introduction

ATAC-seq¹^,² is a robust method that enables identification of regulatory³ open chromatin regions and nucleosome positioning. This information is applied for inferring the location, identity, and activity of transcription factors. The method's sensitivity for measuring quantitative variations in chromatin structure allows the study of the activity of chromatin factors, including chromatin remodelers and modifiers, as well as the transcriptional activity of RNA polymerase II¹. Thus ATAC-seq provides a powerful and unbiased approach for deciphering mechanisms th....