Rapid and Low-Input Profiling of Histone Marks in Plants Using Nucleus CUT&Tag.

Weizhi Ouyang,Guoliang Li,Qing Zhang,Xingwang Li,Xiwen Zhang,Yong Peng,Zhilin Cao

doi:10.3389/fpls.2021.634679

Abstract

Characterizing genome-wide histone posttranscriptional modifications and transcriptional factor occupancy is crucial for deciphering their biological functions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful method for genome-wide profiling of histone modifications and transcriptional factor-binding sites. However, the current ChIP-seq experimental procedure in plants requires significant material and several days for completion. CUT&Tag is an alternative method of ChIP-seq for low-sample and single-cell epigenomic profiling using protein A-Tn5 transposase fusion proteins (PAT). In this study, we developed a nucleus CUT&Tag (nCUT&Tag) protocol based on the live-cell CUT&Tag technology. Our results indicate that nCUT&Tag could be used for histone modifications profiling in both monocot rice and dicot rapeseed using crosslinked or fresh tissues. In addition, both active and repressive histone marks such as H3K4me3 and H3K9me2 can be identified using our nCUT&Tag. More importantly, all the steps in nCUT&Tag can be finished in only 1 day, and the assay can be performed with as little as 0.01 g of plant tissue as starting materials. Therefore, our results demonstrate that nCUT&Tag is an efficient alternative strategy for plant epigenomic studies.

Highlights

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is an efficient method for profiling histone modifications and transcription factor-binding sites (Johnson et al, 2007)
Our results showed that nCUT&Tag is an alternative strategy of ChIP-seq for fast and low-input profiling both active and repressive histone marks with crosslinked or fresh tissues from the monocots or dicots
The nuclei are isolated by centrifuging the filtrate in a swinging bucket rotor at 1000 g for 10 min at 4◦C

Summary

Introduction

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is an efficient method for profiling histone modifications and transcription factor-binding sites (Johnson et al, 2007). In the standard ChIP-seq assay for plants (Kaufmann et al, 2010), formaldehyde-fixed nuclei are isolated and sonicated. Thereafter, the fragmented chromatin is prepared for immunoprecipitation and the ChIP DNA is purified and fragmented for sequencing library preparation. The standard plant ChIPseq assays are complex, requiring large numbers of input cells/tissues and lasting several days from sample fixation to the sequencing-ready library. To improve chromatin profiling efficiency and save experiment time, Zhao et al (2020) developed an enhanced ChIP-seq (eChIP-seq) protocol nCUT&Tag for Efficient Chromatin Profiling with modifications to the standard ChIP-seq. Young leaves of 21-day-old seq, the homogenate chromatin lysates are directly sonicated 2063A seedlings were harvested and crosslinked with 1%

Methods

Results

Conclusion