METHOD AND KIT FOR LIBRARY PREPARATION FROM LOW INPUT CHROMATIN SAMPLES

CONTACT: Jane Luht, University of Tartu, Head of Technology Transfer, jane.luht@ut.ee, Phone: +372 737 4804


BACKGROUND

Chromatin immunoprecipitation (ChIP) has been the method of choice to study the location of DNA bound proteins for years. Coupling ChIP with deep sequencing (ChIP-seq) has enabled to determine the localization of chromatin bound proteins at a genome-wide level. Unfortunately, most of the ChIP-seq methods require millions of cells. It is a huge drawback when working with small cell samples such as adult stem cells or cancer samples from patients.

More sensitive methods have recently emerged, however they are still requiring too much cells (thousands) or need specific lab-on-a-chip instruments and microfluidic solutions. Moreover, those methods are too complex, quite laborious, and expensive.

There is thus still a strong need for new, affordable, simple ChIP-seq methods, truly effectives on small cell samples.


Technology Description

The method of the invention allows to perform high quality chromatin immunoprecipitation (ChIP) coupled with next generation sequencing (ChIP-seq) on samples comprising less than 100 cells. The success of this ChIP-seq method comes from careful integration of different steps in a single tube, ensuring minimal loss of material. The method relies mainly on the following steps: digestion of the chromatin by restriction enzymes, immunoprecipitation of the resulting chromatin fragments, tagmentation of the immobilized chromatin fragments, and amplification (PCR) of the tagmented chromatin fragments.

This new method is designed to work in a single assay tube with no sonication, no crosslinking/decrosslinking steps, and no DNA purification before the amplification step.

It has been successfully validated on histone variants of bovine blastocyst stage embryos as well as on cancer cell lines.


Commercial Applications

This ChIP-seq method allows to study the location, the abundance and the modifications of chromatin bound proteins such as histones or transcription factors at a genome-wide level using very low amount of starting material.

The method is therefore of great interest for the selection of mammalian pre-implantation embryos, in particular at early developmental stages. It also allows studying populations of rare cells such as adult stem cells or cancer cells, in particular at early stages.

More broadly, the ChIP-seq method of the invention can be used to analyze the epigenetic profiles of human clinical samples such as biopsies and blood draws that generally comprise a limited number of cells. Finally, the method of the invention is also applicable to the study of single small animals such as C. elegans or the zebrafish.


Unique Features

The ChIP-seq method of the invention presents unique features that underline its industrial potential:

  • It is a simple and easy to do one tube method
  • It is a time saving method, only one day is necessary to perform it
  • It is an economic method, less chemicals and material are needed, no specific apparatus is needed (such as a sonicator)
  • The method provides high quality results when compared with reference methods
  • The method is very sensitive, samples of less than 100 cells have been successfully analyzed

Intellectual Property Status

A PCT patent application has been filed.


LICENSE

The University of Tartu is looking for partners to develop kits according to the invention. We offer licenses for the method and the kit.