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Femto-seq: Targeted photo-biotinylation, pulldown and sequencing of locus and region-specific DNA from femtoliter volumes within individual cells

Award Number: R01GM145934
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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Femto-seq: Targeted photo-biotinylation, pulldown and sequencing of locus and region-specific DNA from femtoliter volumes within individual cells - Changes in the spatial organization of the genome are directly involved in gene regulation during differentiation, cellular stress responses and disease initiation. Existing approaches such as chromosome conformation capture (3C) methods and DNA fluorescent in situ hybridization (DNA-FISH) have provided information on the overall 3D structure of the nucleus at the chromatin level, providing critical insights into how our genomes are regulated. Nonetheless, new technologies are needed to uncover the finer details on the role of nuclear architecture, topological domains, and genomic interactions. Studies of gene regulation would benefit from a technology that fills the niche between the ensemble averaged 3C methods and the single cell, low throughput DNA-FISH approach. We have recently developed a novel optical technology we call “Femto-seq” that does just this – it allows users to obtain DNA sequence information from targeted femtoliter volumes within the nucleus of selected cells. Femto-seq provides a new way to examine genomic contacts near a specific gene locus or any nuclear region of interest (e.g. nuclear bodies). Using 3D localized two-photon excitation, we can photochemically biotinylate any region of the nucleus we can fluorescently label and identify in volumes which can be as small as a ½ of a femtoliter. The process is carried out on a population of cells using a combined two-photon/confocal microscope which images, locates fluorescently labeled regions of interest and then irradiates those regions using 700 nm femtosecond pulses to biotinylate the chromatin by photochemically cross-linking the DNA with a psoralen-biotin compound. Nuclei are isolated and the biotinylated DNA from the targeted region pulled down and sequenced. Because the cells are imaged to locate the regions of interest, they can also be screened for other parameters, allowing for the collection of targeted biotinylated DNA only from user selected cells within the cell population, providing single-cell like genomic information from a sub-set of cells within the population. We have proof-of-concept data from a cell line with a fluorescently labeled transgene we used as our targeted region, and show that we can obtain DNA highly enriched in transgene locus sequences. The goals of this Focused Technology Research and Development R01 project are to (Aim 1) design and construct a dual confocal/two- photon microscope capable of targeting and irradiating user selected regions-of-interest in a population of cells in a high-throughput automated fashion, (Aim 2) create a chromatin isolation pipeline based on novel microfluidic designs to efficiently purify and prepare the DNA for sequencing, and (Aim 3) demonstrate the improvement obtained from aims 1 and 2 in a series of Femto-seq experiments designed to produce quantitative metrics of improvement and to uncover new knowledge on how environmental signals may be relayed through the cytosol and into the nucleus. Femto-seq is a unique new way of investigating the spatial and regulatory relationships between DNA sequences and any microscopically visible region-of-interest in the nucleus.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $288,008 )
2025	2025	CORNELL UNIVERSITY	341 PINE TREE RD	ITHACA	NY	14850	TOMPKINS	USA	Biomedical Research and Research Training	000	3	1/15/2025	NON-COMPETING CONTINUATION	$288,008
														Subtotal = $288,008

Issue Date FY: 2024 ( Subtotal = $317,772 )
2024	2024	CORNELL UNIVERSITY	341 PINE TREE RD	ITHACA	NY	14850	TOMPKINS	USA	Biomedical Research and Research Training	001	2	6/18/2024	NON-COMPETING CONTINUATION	$31,776
2024	2024	CORNELL UNIVERSITY	341 PINE TREE RD	ITHACA	NY	14850	TOMPKINS	USA	Biomedical Research and Research Training	000	2	1/18/2024	NON-COMPETING CONTINUATION	$285,996
														Subtotal = $317,772

Issue Date FY: 2023 ( Subtotal = $421,283 )
2023	2023	CORNELL UNIVERSITY	341 PINE TREE RD	ITHACA	NY	14850	TOMPKINS	USA	Biomedical Research and Research Training	000	1	1/20/2023	NEW	$421,283
														Subtotal = $421,283

Grand Total All Awards = $1,027,063

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Femto-seq: Targeted photo-biotinylation, pulldown and sequencing of locus and region-specific DNA from femtoliter volumes within individual cells

Award Number: R01GM145934

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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