Diagenode

Single cell ATAC-seq grant winners announcement

Background

Diagenode’s scATAC-seq service makes assessing genome-wide chromatin accessibility studies from individual cells accessible to every researcher. Our method is powered by the combination of Bio-Rad’s droplet-based microfluidic and a hyperactive Tn5 transposase that brings adapters and barcodes to the chromatin of individual nuclei. This best-in-class solution supports a better understanding of the behavior of an individual cell in the context of its environment.

Read more about this technology and its benefits


Meet our grant program winners

Stefan Kurtenbach, PhD, University of Miami, Bascom Palmer Eye Institute, USA

Goal:

Uveal Melanoma is a highly deadly cancer, for which no effective treatment exists. Being in the exceptional position of having access to fresh tumor samples of this rare tumor type directly from the operating room, we have used single cell sequencing, ChIP-seq, RNA-seq, ATAC-seq, methylation profiling and other methods to gain insight into what makes this tumor so deadly. For these projects we utilize various Diagenode products (Bioruptor, tagmentase, antibodies, oligonucleotides, bisulfite conversion kits).

We have recently initiated a clinical trial based on targets we identified from single-cell-sequencing data, which is very promising, but single-cell ATAC-seq data from fresh tumors is urgently needed, to allow us to do transcription factor mapping and identify more novel drug targets. Prime candidates would be verified by ChIP-seq with the Diagenode tagmentase and antibodies.

The challenge with this tumor type is that the cell models available are not good, and bulk ATAC-seq, which we have performed, gets complex due to the presence of immune and other cell populations. This single-cell ATAC-seq award would serve as a stepping stone for bigger grant applications for me as a new PI, and hopefully allow us to initiate more clinical trials to prolong the lives of patients.


DNA solutions:

Single cell ATAC-seq would give us the unique opportunity to identify novel drug targets for this deadly cancer.


Diagenode advantages:

The streamlined process and high capture rate making this technology ideal for larger follow-up studies, keeping data high-quality and reproducible.


Catherine Moermans, PhD, CHU Liege, GIGA, Belgium

Goal:

Asthma is a heterogeneous chronic inflammatory disease that affects an estimated 300 millions of people around the world (https://ginasthma.org/) which represents 5-10% of the population. The severe eosinophilic asthma phenotype is defined according to international guidelines [1] and is linked to a huge healthcare system burden. Anti-IL-5 therapies have emerged as promising therapies for severe eosinophilic asthma. Indeed, Mepolizumab was recently shown to reduce exacerbations and OCS maintenance dose, to improve asthma control and quality of life and to reduce sputum and blood eosinophil counts [2]. However, only 37% of patients reached real asthma control. The aim of this study is to assess in the sputum cells of a cohort of patient before and after treatment with Mepolizumab in order to highlight differences between responders and nonresponders. This project could allow to identify what are the cell types implicated in the treatment response and how they differ from the sputum cells in the non-responder group of patients. Therefore, we will investigate the genome to design chromatin accessibility signatures for each sputum inflammatory cell type for each patient group and then characterize the regulatory landscape of sputum cells.


DNA solutions:

Induced sputum in non-invasive method to obtain cells from airways. It is useful both as a research tool and in the management of asthmatic patients in clinical practice [3]. In addition, induced sputum has been pivotal developing the concept of airway inflammatory phenotype [4]. However, this complex sample is usually contaminated by bacteria and high amounts of RNAses which make them hard to use for gene expression analysis such as RT-qPCR [5]. That is why scATAC-seq could be the method of choice to access the sputum cell genome. Moreover, induced sputum, after the processing which includes a mucolysis step, is a cell suspension composed of a mixture of inflammatory cells (mainly macrophages, neutrophils, eosinophils, lymphocytes and few epithelial cells) [6]. With this kind of heterogeneous sample, bulk technologies may miss important signals coming from a small cell subset. The overview of the genome-wide chromatin accessibility and sequencing of all the cell types, the transcription factors binding sites and DNA methylation sites will give us tones of information in order to differentiate anti-IL-5 responders versus not and understand the mechanisms of antiIL-5 resistance. For this purpose, we will collect the sputum of patients with severe eosinophilic asthma (sputum eosinophil proportion greater than 3%) before and after treatment. We will also collect the demographic characteristics of the patients, the asthma control and quality of life scores, the differential leucocyte count of the blood and sputum and the lung functions results. Sputum samples will be processed in our pneumology lab, including samples before and after anti-IL5 therapy for 2 patients with a treatment response compared to 2 patients without (based on the asthma control and quality of life questionnaires, lung function test results, decrease of blood and sputum eosinophil count). The number of sputum cells is usually few millions/ml which is not a limiting factor. What could be more restrictive is the cell viability and squamous cell contamination by the mouth cells. The cell viability is assessed with trypan blue staining and the squamous cells contamination is reported. The samples with the best parameters will be selected for scATAC-seq. References [1] doi:10.1183/09031936.00202013. [2] doi:10.2174/092986711795328337. [4] doi:10.1186/1471-2466-13-11. [5] doi:10.1165/rcmb.2017-0198LE. [6] doi:10.3791/56612.


Diagenode advantages:

Diagenode has a huge expertise in epigenetics and we need the best guidance through the accomplishment of this project. We have no skills in bio-informatic analysis and ATAC-seq technology data must be analyzed by competent scientists to maximize the quality of the results as this part is a real challenge.



Christian Hedrich, PhD, University of Liverpool, United Kingdom

Goal:

Independent of the patient’s age, it is difficult to achieve an early diagnosis of psoriatic arthritis (PsA) in the absence of biomarkers. Indeed, in up to 50% of patients, arthritis precedes skin changes, thus mimicking other forms of arthritis. Childhood psoriasis presents with particularly severe phenotypes underscoring the need for research in this age group to promote scientific progress through the identification of molecular targets and pathophysiological contributors to disease that can then be tested in larger cohorts.

Hypothesis:

Expansion and activation of innate and adaptive immune cells in psoriasis and psJIA may reflect disease- and outcome-specific events that contribute to inflammation and damage. Defining cellular and molecular patterns will aid in diagnosing patients at an early stage, and define new diagnostic and prognostic biomarkers, and therapeutic targets.In this pilot project, we will ask whether immune cell subpopulations represent:i) aberrantly activated cells (monocytes, pDCs).


DNA solutions:

Single cell ATAC sequencing will allow epigenetic profiling of small cell numbers, including rare immune cell subsets in bodily fluids (blood and synovial fluids) of patients with childhood psoriatic arthritis and controls. Single cell data from PBMCS from controls (N=4) will be compared to PBMCS from patients with psoriatic arthritis and immune cells from synovial fluid of the same patients (N=3).As overall cell numbers available from synovial fluid (but in children also blood) are limited and include rare cell subsets that may centrally contribute to disease pathogenesis and phenotypes, single cell ATA sequencing is the method of choice to generate reliable and biologically meaningful epigenetic data. Single cell technology will allow cell type identification and comparison of epigenetic patterns between controls vs patients, and between sample sources (blood vs synovial fluid).Additional cells collected from the same patients at the same time will be used towards RNA expression profiling using single-cell ATAC-seq.


Diagenode advantages:

BioRad SureCell technology offers robust and sensitive analysis of small cell numbers at the single-cell level. Provided small sample sizes available from pediatric patients, especially investigating synovial fluid immune cells, this offers a big and invaluable advantage when compared to other technologies available. In our case, this will allow mRNA expression profiling from additional cells that remain for RNA sequencing. Diagenode offers reliable and reproducible data based on highest quality standards and vast experience in epigenetic profiling.


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