Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag

PLoS One. 2021 Nov 16;16(11):e0259846. doi: 10.1371/journal.pone.0259846. eCollection 2021.

Abstract

Epigenome research has employed various methods to identify the genomic location of proteins of interest, such as transcription factors and histone modifications. A recently established method called CUT&Tag uses a Protein-A Tn5 transposase fusion protein, which cuts the genome and inserts adapter sequences nearby the target protein. Throughout most of the CUT&Tag procedure, cells are held on concanavalin A (con A)-conjugated magnetic beads. Proper holding of cells would be decisive for the accessibility of Tn5 to the chromatin, and efficacy of the procedure of washing cells. However, BioMag®Plus ConA magnetic beads, used in the original CUT&Tag protocol, often exhibit poor suspendability and severe aggregation. Here, we compared the BioMag beads and Dynabeads® magnetic particles of which conjugation of con A was done by our hands, and examined the performance of these magnetic beads in CUT&Tag. Among tested, one of the Dynabeads, MyOne-T1, kept excessive suspendability in a buffer even after overnight incubation. Furthermore, the MyOne-T1 beads notably improved the sensitivity in CUT&Tag assay for H3K4me3. In conclusion, the arrangement and the selection of MyOne-T1 refine the suspendability of beads, which improves the association of chromatin with Tn5, which enhances the sensitivity in CUT&Tag assay.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Concanavalin A / administration & dosage*
  • Concanavalin A / chemistry
  • Concanavalin A / pharmacology
  • Epigenomics
  • HEK293 Cells
  • Histone Code
  • Histones / metabolism*
  • Humans
  • Immunomagnetic Separation
  • Magnetic Fields
  • Methylation
  • Mice
  • Particle Size
  • Recombinant Fusion Proteins / metabolism
  • Staphylococcal Protein A / genetics*
  • Staphylococcal Protein A / metabolism
  • Transposases / genetics*
  • Transposases / metabolism

Substances

  • Histones
  • Recombinant Fusion Proteins
  • Staphylococcal Protein A
  • Tn5 transposase
  • Concanavalin A
  • Transposases

Grants and funding

This work was supported in part by JSPS KAKENHI grant number 17K15392 and 20H00446 (to Y.F.) and JST ERATO (JPMJER1901 to Y.O.). Y.T. is employed by VERITAS corporation. The funder provided support in the form of salaries for Y.T., but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of Y.T. are articulated in the ‘author contributions’ section.