摘要Diffuse midline glioma(DMG),H3K27-altered,is lethal pediatric-type,high-grade,localized to the midline region of the central nervous system.Effective treatment guidelines are absent,and clinical trials are preferred for primary or recur-rent DMG patients.Recently,epigenetic agent-based immunotherapy has exhibited promising therapeutic effects in the clinical setting.However,the underlying mechanisms remain a mystery.The rare DMG tumor samples from biopsy or resection largely impede basic research,by using patient-derived tumor cells which better recapitulate the parental tu-mor's heterogeneity compared to established cell lines.As an epigenetic reprogramming disease,DMG exhibits a global loss of H3K27 trimethylation(H3K27me3)and a gain of H3K27 acetylation(H3K27ac).Analysis of multiple epige-netic marks is fundamentally necessary.However,traditional techniques cannot allow ultra-low input and high-throughput.Herein we have developed a new method called high-throughput in situ tagged immunoprecipitation sequencing(HiTIP-seq),which uses an integrated superhydrophobic microwell array technology(InSMART).We were able to perform 100 parallel assays from as few as 100 cells per microwell on a single chip.We applied the tech-nology to profile epigenetic alterations of three-dimensional(3D)cell cultures derived from DMG patients.Our HiTIP-seq integrated with RNA sequencing(RNA-seq)analysis revealed that the combination of epigenetic agents(panobino-stat and tazemetostat),reprogrammed histone modifications and drove transcriptome changes.Among them,Wnt inhibitory factor 1(WIF1)has a gain of H3K27ac and a loss of H3K27me3,which leads to the upregulated expression.Altogether,HiTIP-seq is a versatile method for high-throughput analysis of histone modifications,suitable for both DMG research and studying rare 3D models.