Fig. 4: Single-nuclei ATAC, RNA, and CRISPRi perturbation sequencing identify integrated genomic mechanisms driving schwannoma cell state evolution.

a Experimental workflow for single-nuclei ATAC, RNA, and CRISPRi perturbation sequencing (snARC-seq). Triplicate HEI-193 cultures were transduced with sgRNA libraries targeting 29 epigenetic regulators driving radiotherapy responses from genome-wide CRISPRi screens (Fig. 3c) and treated with 0 Gy or 1.8 Gy × 5 of radiotherapy prior to isolation of single-nuclei for sequencing. sgRNA identities were recovered from CROP-seq tags in single-nuclei RNA sequencing data. b UMAPs of ATAC (left) or RNA (right) sequencing of 855 single nuclei passing snARC-seq quality control from triplicate control or radiotherapy conditions (Supplementary Fig. 18). c Hierarchical clustering of differential gene activity scores between radiotherapy and control conditions for each snARC-seq perturbation (columns). Gene activity modules (rows) were derived from HEI-193 schwannoma cell states ± radiotherapy (Fig. 2c) or from human schwannoma cell types (Fig. 1b). Gene ontology of perturbed epigenetic regulators and CRISPRi screen growth (gamma) or radiation response (rho) phenotypes from genome-wide CRISPRi screens (Fig. 3c) are shown. d Hierarchical clustering of differential ChromVAR transcription factor motif deviations between radiotherapy and control conditions for each snARC-seq perturbation (columns). e Average profile plots of normalized ATAC signal at KLF13 or TCF3 motifs with ENCODE ChIP-seq peak annotations and differential accessibility following snARC-seq perturbation of KDM1A, KDM5C, or SETDB1. f Feature plot of integrated UMAP from harmonized schwannoma single-nuclei and single-cell RNA sequencing (Fig. 1b) showing genes near TCF3 motifs that are differentially accessible following SETDB1 snARC-seq perturbation. g Hierarchical clustering of human schwannoma RNA sequencing profiles using 56 differentially expressed SETDB1 targets with TCF3 motifs showing separation of NCS and IES molecular groups of schwannomas. Source data are provided as a Source Data file.