OPCs constitute 5 to 8% of the total number of cells in the adult central nervous system (CNS) being the main population that generates OLs during CNS development and remyelination events. The specification of OPCs occurs in different waves and from different domains at the ventricles of the developing brain and spinal cord. OLs produce myelin, a lipid-rich membrane that insulates neuronal axons. Several diseases, such as MS, are characterized by abnormal or defective myelination. Spontaneous remyelination occurs at initial stages of MS, promoted by endogenous OPCs. However, this process progressively starts occurring with less efficiency, until it eventually fails. In order to achieve remyelination processes in the context of MS, the population to target in a therapeutic context are the OPCs. In order to characterize the precursor and OL lineage development through single cell transcriptomics and epigenomics in disease and healthy states, our group will perform sequencing of nuclei single-cell from postmortem brain material from MS patients and healthy controls using droplet scRNA-Seq (Chromium 10x Genomics). We have already sequenced 20 samples of MS and Controls patients, as the start of the project. We will also include human fresh brain samples from patients with hydrocephaly from both genders from a range of age of 60-70 years old. The collected samples belong to the frontal cortex, specifically from the Kocher region. The surgery consists in a non-invasive procedure where we will collect otherwise discarded tissue. Samples will be sequenced using single cell RNA-Seq technologies, including 10x Genomics droplet sequencing (Chromium). We have scheduled around 4 surgeries per month. From which we are planning to single sequence when the tissue allows. We will also perform single-cell RNA-Seq to characterize human oligodendrocyte lineage cells obtained from human fetal (7-11 weeks) forebrain CNS tissue (collaboration with Dr. Erik Sundström, Karolinska Institutet). From these samples, we are also planning to applying epigenomic tehcnologies, for instance to study the chromatin accessible with bulk and single cell ATAC-Seq and also the 3D chromatin architecture, using GAM. In order to allow the spatial mapping of the specific regulatory features involved in OL development in human, we will generate genome architecture mapping (GAM) data. GAM is a novel methodology that allows mapping genome wide chromatin interactions by cryosectioning slices randomly from nuclei. This work involves sensitive personal data, which will require a secure storage and computing server space. Requested resources Chromium 10x Genomics raw fastq data ~ 2.GB + 2.5GB of CellRanger processed outs , per sample scATAC-seq, bulkATAC-seq raw fastq files ~ 2GB per sample Current data: 345.8 GB of raw sequencing data 125.1 GB nuclei 1 31.9 nuclei 2 9.9 GB fresh nuclei 20 GB MS nuclei SS2 57.4 GB fetal 1 101.5 GB fetal 2