Abstract
INTRODUCTION: Recent evidence suggests epigenetic heterogeneity may be the primary driver of cell phenotype and treatment resistance in glioblastoma (GBM). Epigenetic heterogeneity is defined by alterations of chromatin histone modifications with activating marks such as H3K27 acetylation and repressive marks such as H3K27 trimethylation. Combinations of these histone marks govern the activity of functional genomic elements such as enhancers or promoters. Single-cell (“sc-”) technologies have only recently been adapted to the study of epigenetic cell states and significant technical challenges have limited their widespread adoption, especially in complex tissues. Here, we report the development of an optimized method for performing tissue processing and simultaneous single-cell epigenetic and transcriptomic analyses in flash frozen GBM samples. METHODS: Building on our prior work, we developed a new protocol, multiomic single-cell cleavage-under-targets and tagmentation, or “mscCUT&TAG”. We optimized nuclei isolation, cleanup and permeabilization from flash frozen GBM and reaction conditions for the scCUT&TAG portion of the assay. Critically, these changes maintained high-quality, intact nuclei in suspension while limiting well-described technical issues such as nuclei clumping and sample loss. Furthermore, we incorporated the ability to multiplex both patient samples and histone marks. RESULTS: We applied 10X scRNA-seq, 10X Multiome (RNA+ATAC-seq), and mscCUT&TAG for five histone marks to the same GBM sample, enabling an integrated evaluation of the tumor’s transcriptome, chromatin accessibility and epigenetic regulation at single-cell resolution. mscCUT&TAG enabled the first assessment of single-cell genome segmentation in GBM. We integrated across modalities to identify specific tumor cell populations, cell-type specific coordinated changes in epigenetics and transcriptome, and define functional regulatory elements. CONCLUSIONS: We developed a novel multiomic protocol, mscCUT&TAG, that allows for a comprehensive analysis of epigenetic heterogeneity in GBM. We anticipate this protocol will enhance our understanding of fundamental biology and potential therapeutic avenues in GBM.