A reoccurring issue in neuroepigenomic studies, especially in the context of neurodegenerative disease, is the use of (heterogeneous) bulk tissue, which generates noise during epigenetic profiling. A workable solution to this issue is to quantify epigenetic patterns in individually isolated neuronal cells using laser capture microdissection (LCM). For this purpose, we established a novel approach for targeted DNA methylation profiling of individual genes that relies on a combination of LCM and limiting dilution bisulfite pyrosequencing (LDBSP). Using this approach, we determined cytosine-phosphate-guanine (CpG) methylation rates of single alleles derived from 50 neurons that were isolated from unfixed post-mortem brain tissue. In the present manuscript, we describe the general workflow and, as a showcase, demonstrate how targeted methylation analysis of various genes, in this case, RHBDF2, OXT, TNXB, DNAJB13, PGLYRP1, C3, and LMX1B, can be performed simultaneously. By doing so, we describe an adapted data analysis pipeline for LDBSP, allowing one to include and correct CpG methylation rates derived from multi-allele reactions. In addition, we show that the efficiency of LDBSP on DNA derived from LCM neurons is similar to the efficiency obtained in previously published studies using this technique on other cell types. Overall, the method described here provides the user with a more accurate estimation of the DNA methylation status of each target gene in the analyzed cell pools, thereby adding further validity to this approach.

Background: A challenge frequently encountered in neuroepigenomic research, especially in the context of neurodegenerative diseases such as Alzheimer’s disease, is the difficulty of measuring epigenetic patterns in heterogenous bulk tissue samples, as these are known to cause noise during profiling. A viable strategy to overcome this challenge is to analyze epigenetic marks in single neurons that have been isolated using laser capture microdissection (LCM).Method: In this study, we developed a new approach for targeted DNA methylation profiling of individual genes that combines the use of LCM and limiting dilution bisulfite pyrosequencing (LDBSP). By utilizing this method, we were able to determine the cytosine‐phosphate‐guanine (CpG) methylation rates of single alleles obtained from 50 neurons, which were isolated from unfixed post‐mortem brain tissue.Result: We demonstrate how the methylation status of multiple genes such as RHBDF2, OXT, TNXB, DNAJB13, PGLYRP1, C3, and LMX1B, can be quantified simultaneously using the method described above. In addition, we provide an adapted data analysis pipeline for LDBSP, which accounts for and corrects methylation rates obtained from multi‐allele reactions. Furthermore, we show that the efficiency of LDBSP on DNA derived from LCM‐isolated neurons is comparable to the efficiency in previous studies using other cell types.Conclusion: This method allows for a more precise determination of the DNA methylation status of each targeted gene in the neuronal cell samples under investigation, thereby increasing the validity of the technique.