We used different colors to indicate the TE information at the integration site. 8). Strikingly, a significant quantity of CAR-T integration events occurred at genomic regions that were inaccessible across the populace of cells. KRX-0402 In addition to charting the chromatin convenience state of host genome, EpiVIA was also able to detect the convenience state of the viral genome at the single-cell resolution. Because the standard analysis of bulk and KRX-0402 scATAC-seq datasets reveals several layers of cell identity, including the unbiased identification of regulatory elements (9), inference of transcription factor binding sites (10, 11), and nucleosome positions (12), we anticipate that EpiVIAs addition of retroviral integration site analysis to this multifaceted assay should enable discovering cellular fates associated with durable CAR-T treatment. Results Reconstructing Lentiviral Integration Sites from Chromatin Convenience Measurements Using EpiVIA. We postulated that this transposase used in the ATAC-seq protocol can also fragment the proviral genome, and that the paired-end sequencing of such fragments followed by aligning the reads to host and viral genomes can delineate the precise location of lentiviral integration events (Fig. 1(13), a BurrowsCWheeler aligner, which is usually capable of mapping paired-end reads to two unique chromosomes. In a combined host-viral genome, five possibilities exist for mapping the two ends of a fragment: (case A) Mapping of both ends to the host genome, (case B) mapping of both ends to the viral genome, (case C) mapping of one end to the viral genome and the other end to the host genome (referred to as pair-chimeric), (case D) mapping of one end to the host genome and the other end to the host and viral genomes (referred to as host-chimeric), and (case E) mapping of one end to the viral genome and the other end to both host and viral genomes (referred to as viral-chimeric) (Fig. 1and allele KRX-0402 disrupted the function of the gene. Ultimately, this patient went into remission because of the clonal growth of a single CAR-T cell and has remained cancer free in the 6 y since, with CAR-T cells derived from this single clone still circulating in his peripheral blood (16). We examined the ability of EpiVIA to determine CAR-T integration sites in CAR+ CD8+ T cells sorted from this patient and used his CAR? CD8+ T cells as a negative control (16). We found the selective enrichment of host-viral chimeric reads at the LTRs of the lentiviral genome in CAR+ but not CAR? T cells, corroborating the integration of the provirus in CAR+ T cells (Fig. 2 and and the host genome (Fig. 2gene in addition to demarcating chromatin convenience at the site of integration. Altogether, using multiple clonal contexts with predefined integration sites, we exhibited that EpiVIA can reliably detect lentiviral integration events in addition to mapping the chromatin convenience state of the entire genome at the population level. EpiVIA Can Detect CAR-T NES KRX-0402 Integration Sites at the Single-Cell Level. To investigate whether EpiVIA can link cell identity and CAR-T integration sites at the single-cell resolution, we mapped chromatin convenience using scATAC-seq KRX-0402 in droplets exploiting the commercially available Chromium platform (10X Genomics) for 5,000 human CD8+ T cells. Bulk human T cells from a healthy donor were isolated and activated in vitro with CD3/CD28 Dynabeads and high-dose IL-2 for 24 h, followed by transduction with the CAR lentivirus. The cells were then expanded over the course of 9 days. To ensure the expression of CAR and CD8 proteins, cells were further purified using magnetic beads. Sequencing of scATAC-seq libraries generated 744,921,436 read pairs and the key summary metrics from your Cell Ranger pipeline suggested high quality of single-cell chromatin convenience data in CAR+ CD8+ T cells (and or genes (repeats (Fig. 3 values were calculated by Fishers exact test, followed by FDR correction. (value calculated with Wilcoxon rank-sum test. (locus with CAR-T integration sites recognized by EpiVIA in two single CAR-T cells together with scATAC-seq data across all single.