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Dna-barcoded nucleosomes for chromatin mapping assays

A nucleosome and barcoding technology, applied in the field of DNA barcoding recombinant nucleosomes, can solve the problem of incompatibility of immune tethering methods

Pending Publication Date: 2021-10-15
EPICYPHER INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current "ChIP compatible" DNA barcoded recombinant nucleosomes are not compatible with chromatin accessibility assays (e.g. MNase-seq or ATAC-seq) and immunotethering methods (e.g. CUT&RUN or CUT&Tag)

Method used

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  • Dna-barcoded nucleosomes for chromatin mapping assays
  • Dna-barcoded nucleosomes for chromatin mapping assays
  • Dna-barcoded nucleosomes for chromatin mapping assays

Examples

Experimental program
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Embodiment 1

[0232] Example 1: Schematic of how DNA barcoded nucleosomes can be used for chromatin accessibility and chromatin mapping assays.

[0233] The following example shows how DNA-barcoded incorporation into nucleosomes can be used as an incorporation control in genome mapping assays using solid supports. Of note, nucleosome incorporation can also be used in a similar workflow without the use of solid supports. exist figure 1 In the example above, the ATAC-seq workflow is shown, where nucleosome incorporation can be added to the sample (cells or nuclei) either before or after the sample is bound to the solid support. After fixation, samples are permeabilized and incubated with Tn5 (loaded with adapters compatible with Illumina sequencers), which binds to open chromatin. Labeling is initiated by adding magnesium, followed by DNA purification, PCR, and next-generation sequencing. Similar to sample chromatin, Tn5 will bind and label adapter DNA incorporated on nucleosomes. DNA rec...

Embodiment 2

[0234] Example 2: Development of DNA barcoded nucleosome incorporation controls for CUT & RUN assays.

[0235] Various DNA configurations can be used as nucleosome positioning sequences to develop DNA barcoded recombinant nucleosome incorporations compatible with immunotethering methods such as CUT&RUN or CUT&Tag. Here, two different DNA templates for nucleosome assembly are shown. Each template was derived from the Widom 601 sequence (Lowary and Widom 1998) and modified to include a 24bp barcode near the 147bp 3' end. The 147bp nucleosome positioning sequence was also flanked by 5' biotin and a linker of less length ( Figure 2A-2B ). Using these templates next, a pilot panel of DNA barcoded nucleosomes was generated using recombinantly modified octamers that were either unmodified or carried H3K4me1, H3K4me2, or H3K4me3 (each nucleosome consisted of a unique DNA barcode representation; Figure 2C ). Next, it was tested whether these nucleosomes could be used to monitor ...

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Abstract

The present invention relates to DNA-barcoded recombinant nucleosomes and polynucleosomes that have been engineered for use as spike-in controls for chromatin accessibility assays, chromatin mapping assays, e.g., using tethered enzymes, as well as other chromatin assays. The invention further relates to methods of using the engineered DNA-barcoded recombinant nucleosomes in chromatin accessibility assays, chromatin mapping assays, as well as other chromatin assays.

Description

[0001] priority statement [0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 783,861, filed December 21, 2018, the entire contents of which are incorporated herein by reference. [0003] field of invention [0004] The present invention relates to DNA barcoded recombinant nucleosomes and polynucleosomes that have been engineered (e.g., using tethered enzymes) for use in chromatin accessibility assays, chromatin mapping assays, and other chromatin assays The incorporation control. The invention also relates to methods of using engineered DNA barcoded recombinant nucleosomes in chromatin accessibility assays, chromatin mapping assays, and other chromatin assays. [0005] Background of the invention [0006] Chromatin structure drives regulation of gene transcription by controlling accessibility to regulatory mechanisms that engage DNA, such as transcription factors (Stavreva and Hager 2015). This process is partly controlled by the local...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6874C12Q1/6806
CPCC12Q1/6806G01N2500/10C12Q2563/131C12Q2563/179C12Q2565/133C12Q2565/514G01N33/6875
Inventor M·W·考尔斯Z-W·孙M-C·基奥R·J·文特雷斯E·N·温扎普菲
Owner EPICYPHER INC