Single-Molecule Multikilobase-Scale Profiling of Chromatin Accessibility Using m6A-SMAC-Seq and m6A-CpG-GpC-SMAC-Seq

Georgi K Marinov; Zohar Shipony; Anshul Kundaje; William J Greenleaf

doi:10.1007/978-1-0716-2140-0_15

Single-Molecule Multikilobase-Scale Profiling of Chromatin Accessibility Using m6A-SMAC-Seq and m6A-CpG-GpC-SMAC-Seq

Methods Mol Biol. 2022:2458:269-298. doi: 10.1007/978-1-0716-2140-0_15.

Authors

Georgi K Marinov^#¹, Zohar Shipony^#², Anshul Kundaje^{3

4}, William J Greenleaf^{3

5

6

7}

Affiliations

¹ Department of Genetics, Stanford University, Stanford, CA, USA. marinovg@stanford.edu.
² Department of Genetics, Stanford University, Stanford, CA, USA. zshipony@stanford.edu.
³ Department of Genetics, Stanford University, Stanford, CA, USA.
⁴ Department of Computer Science, Stanford University, Stanford, CA, USA.
⁵ Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA, USA.
⁶ Department of Applied Physics, Stanford University, Stanford, CA, USA.
⁷ Chan Zuckerberg Biohub, San Francisco, CA, USA.

^# Contributed equally.

Abstract

A hallmark feature of active cis-regulatory elements (CREs) in eukaryotes is their nucleosomal depletion and, accordingly, higher accessibility to enzymatic treatment. This property has been the basis of a number of sequencing-based assays for genome-wide identification and tracking the activity of CREs across different biological conditions, such as DNAse-seq, ATAC-seq , NOMeseq, and others. However, the fragmentation of DNA inherent to many of these assays and the limited read length of short-read sequencing platforms have so far not allowed the simultaneous measurement of the chromatin accessibility state of CREs located distally from each other. The combination of labeling accessible DNA with DNA modifications and nanopore sequencing has made it possible to develop such assays. Here, we provide a detailed protocol for carrying out the SMAC-seq assay (Single-Molecule long-read Accessible Chromatin mapping sequencing), in its m6A-SMAC-seq and m6A-CpG-GpC-SMAC-seq variants, together with methods for data processing and analysis, and discuss key experimental and analytical considerations for working with SMAC-seq datasets.

Keywords: Chromatin accessibility; DNA modifications; EcoGII; Nanopore sequencing; SMAC-seq; m6A.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Chromatin Immunoprecipitation Sequencing
Chromatin* / genetics
High-Throughput Nucleotide Sequencing* / methods
Nucleosomes
Sequence Analysis, DNA / methods

Substances

Chromatin
Nucleosomes

Abstract

Publication types

MeSH terms

Substances

Grants and funding