Using Cleavage Under Targets and Tagmentation (CUT&Tag) Assay in Mouse Myoblast Research

Summary

Researchers new to the epigenetic field will find CUT&Tag a significantly easier alternative to ChIP assays. CUT&Tag has tremendously benefited the epigenetic studies on rare and primary cell populations, generating high-quality data from very few cells. This protocol describes performing H3K4me1 CUT&Tag assays on mouse myoblasts isolated from mouse hindlimb muscles.

Abstract

This protocol paper aims to provide the new researchers with the full details of using Cleavage Under Targets and Tagmentation (CUT&Tag) to profile the genomic locations of chromatin binding factors, histone marks, and histone variants. CUT&Tag protocols function very well with mouse myoblasts and freshly isolated muscle stem cells (MuSCs). They can easily be applied to many other cell types as long as the cells can be immobilized by Concanavalin-A beads. Compared to CUT&Tag, chromatin immunoprecipitation (ChIP) assays are time-consuming experiments. ChIP assays require the pre-treatment of chromatin before the chromatic material can be used for immunoprecipitation. In cross-linking ChIP (X-ChIP), pre-treatment of chromatin involves cross-linking and sonication to fragment the chromatin. In the case of native ChIP (N-ChIP), the fragmented chromatins are normally achieved by Micrococcal nuclease (MNase) digestion. Both sonication and MNase digestion introduce some bias to the ChIP experiments. CUT&Tag assays can be finished within fewer steps and require much fewer cells compared to ChIPs but provide more unbiased information on transcription factors or histone marks at various genomic locations. CUT&Tag can function with as few as 5,000 cells. Due to its higher sensitivity and lower background signal than ChIPs, researchers can expect to obtain reliable peak data from merely several millions of reads after sequencing.

Introduction

CUT&Tag assay was invented to compensate for some overt flaws of ChIPs¹. The two major disadvantages of ChIPs are 1) the bias introduced when fragmenting chromatin and 2) the incompetence to work with low cell numbers. X-ChIP assays rely on either sonication or MNase digestion to get chromatin fragments, whereas N-ChIP mostly uses MNase digestion to get nucleosomes. Sonication shows a bias towards relaxed chromatin locations such as promoter regions², and apparently, MNase digestion also works more efficiently on relaxed chromatin fibers. Moreover, some reported that MNase digestion also shows a DNA sequence-dependen....

Protocol

The methods presented in this manuscript are all approved by the Institutional Animal Care and Use Committee of Guangzhou Laboratory. Mice used to generate this manuscript's representative results were housed and maintained in accordance with the guidelines of the Institutional Animal Care and Use Committee of Guangzhou Laboratory.

1. Myoblast isolation from mouse hindlimb muscles (Example of using 1 mouse)

1. Dilute glacial acetic acid with ddH₂O to 0.02 M and autoclave it.
2. Dilute collagen (from rat tail) to 0.1 mg/mL with 0.02 M acetic acid.
3. Add this collagen solution into cultu....

Results

Before binding cells to Concanavalin-A beads, check the cell suspension under the microscope. Accordingly, after incubating the cells with Concanavalin-A beads, put the sample tubes on the magnetic rack, and the supernatant should be again observed using a microscope. This is to assess how efficiently the cells have been captured by Concanavalin-A beads. Wash buffer containing 7 x 10⁵ cells/mL should look like Figure 1A under the microscope. In contrast, Figur.......

Discussion

The specific cell number required in a certain CUT&Tag reaction completely relies on the enrichment of the histone marks/variants or chromatin-binding proteins that are to be tested. Normally for very enriched histone marks such as H3K4me1, H3K4me3, and H3K27ac etc., 25,000-50,000 myoblasts are quite sufficient for one CUT&Tag reaction. However, some rare chromatin-binding proteins might require up to 250,000 cells. The cell number used in CUT&Tag assays is critical, which, if not handled well normally causes.......

Materials

Name	Company	Catalog Number	Comments
bFGF	R&D Systems	233-FB-025
Collagen	Corning	354236
Collagenase II	Worthington	LS004177
Concanavalin-A	Sigma-Aldrich	C5275
Concanavalin-A beads	Bangs Laboratories	BP531
Digitonin	Sigma-Aldrich	300410
Dispase II	Thermo Fisher Scientific	17105041
Fetal bovine serum	Hyclone	SH30396.03
H3K4me1 antibody	abcam	ab8895
Ham's F10 media	Thermo Fisher Scientific	11550043
Hyperactive Universal CUT&Tag Assay Kit for Illumina	Vazyme	TD903	This kit has been tested by us to function well
Magnetic rack for 1.5 mL EP tubes	Qualityard	QYM06
Magnetic rack for 8-PCR tube stripes	Anosun Magnetic	CLJ16/21-021
NEBNext High-Fidelity 2x PCR Master Mix	NEB	M0541L	For library-making PCR reaction
pA-Tn5	Vazyme	S603-01	Needs to be mounted with adaptors before use
Protease inhibitor cocktail	Sigma-Aldrich	5056489001
Proteinase K	Beyotime	ST535-100mg
RPMI-1640 media	Thermo Fisher Scientific	C11875500BT
Secondary antibody (Guinea Pig anti-rabbit IgG)	Antibodies-online	ABIN101961
Spermidine	Sigma-Aldrich	S2626
TruePrep Index Kit V2 for Illumina	Vazyme	TD202	This kit provide Illumina N7XX and N5XX primers
VAHTS DNA Clean Beads	Vazyme	N411	Can substitute Ampure XP beads. Can purify CUT&Tag libraries and select DNA fragments by size