Recap Work Package 5 year 1

Andrew Li Yim and Anje te Velde lead the team in WP5 of the METHYLOMIC project, alongside Wouter de Jonge, Peter Henneman and Femke Mol. In its inaugural year, this team aims to discern whether DNA methylation signals originate from methylation itself or variations in cellular composition. Utilizing advanced single-cell technologies, they target specific DNA methylation patterns unique to various cell types. The following text provides a summary of the first year of research within WP5.

Single-cell technologies

The goal of work package 5 is to understand the origin of the observed DNA methylation signal of the predictor CpGs. Simply put, does the signal originate from actual DNA methylation, or a difference in cellular composition. To this end, we aim to pinpoint specific DNA methylation patterns that are unique to different cell types. To achieve this, we’ve looked for single-cell technologies that are capable of characterizing the cellular composition based on gene expression while simultaneously quantifying the DNA methylome at a single-cell level. Our strategy integrates two methodologies: smartSeq2 for characterizing cell populations via RNA expression, and reduced representation bisulfite sequencing (RRBS) for exploring the DNA methylome.

First step

The first step is to isolate white individual white blood cells from blood samples, followed by the isolation of both DNA and RNA from each cell. Ultimately, this will result in a paired DNA methylome and transcriptome per cell, for which we will develop a novel bioinformatic pipeline to dissect and interpret the data.

We anticipate that one of the challenges lies in the complexity of the blood samples. Blood encompasses different cell types, including erythrocytes, leukocytes, platelets, and plasma. In context of inflammation, our focus narrows down to the leukocyte fraction. Yet, within this subset there are more subdivisions: granulocytes, lymphocytes, and monocytes. Granulocytes are particularly challenging due to their susceptibility to degradation after collection.

Blood samples

We are now getting ready to put our techniques to the test using blood samples from volunteers with the goal of optimizing the protocol necessary to conduct the final analyses on samples obtained from patients that are scheduled to start treatment. We anticipate that our observations will provide insights into the mechanisms by which response manifests, potentially allowing us to refine existing treatments and pave the way for novel therapeutic interventions.

Text: Femke Mol