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The epigenome and transcriptome constitute a critical element of a tightly regulated, cell-type specific gene expression program, and subtle perturbations in the regulation of this program can result in pathology. Epigenetic features such as DNA accessibility dictate transcriptional regulation in a cell type- and cell state- specific manner, and mapping this in health vs. disease in clinically relevant material is opening the door to new mechanistic insights and new targets for therapy. Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) allows profiling of chromatin accessibility with low cell input, making it amenable to the clinical setting, such as peripheral blood from clinical trials, and this makes it applicable to rare cell populations, such as regulatory T (Treg) cells. However, there is little known about the compatibility of the assay on materials recovered from cryopreserved rare cell populations. In the context of tolerance or autoimmunity, regulatory T cells play a critical role in maintaining immune homeostasis, and loss of numbers or function is linked to many diseases, making them a clinically relevant population to analyse using genomic platforms. Here we demonstrate the robustness and reproducibility of an ATAC-seq protocol comparing fresh or cryopreserved primary Treg cells, and comparing their profile in the steady state and in response to stimulation. We extend this method to explore the feasibility of conducting simultaneous quantitation of chromatin accessibility and transcriptome from a single aliquot of 50,000 Treg cells from cryopreserved PBMCs. Profiling of chromatin accessibility and gene expression in parallel within the same pool of cells controls for cellular heterogeneity and will be particularly beneficial for experiments constrained by limited input material, such as biobanked PBMC from clinical trials. This approach will be complementary to single-cell experiments as libraries used to profile chromatin accessibility and transcriptome are derived from the same population of cells, controlling for stochastic gene fluctuation in different cells in a population at any given time. Overall, we observed a high correlation of accessibility patterns and transcription factor (TF) dynamics between fresh Treg cells and cells recovered from cryopreservation samples. The distribution of fragment size, enrichment of transcription start sites (TSS) and genomic features of thawed Treg cells recapitulate that of the fresh cells. Furthermore, highly consistent global chromatin and transcriptional changes in response to stimulation were observed in both fresh and frozen samples. Lastly, highly similar transcriptomic profiles were obtained from whole cells and from the supernatants recovered from ATAC-seq reactions. This report highlights the feasibility of applying these techniques to profile the epigenomic landscape of cells recovered from cryopreservation biorepositories. Implementation of this approach is suitable in biorepositories and will contribute to advances in the field of translational research and personalized medicine.

usc Refereed/Peer-reviewed Purpose: Genetic variants causing aberrant premessenger RNA splicing are increasingly being recognized as causal variants in genetic disorders. In this study, we devise standardized practices for polymerase chain reaction (PCR)-based RNA diagnostics using clinically accessible specimens (blood, fibroblasts, urothelia, biopsy). Methods: A total of 74 families with diverse monogenic conditions (31% prenatal-congenital onset, 47% early childhood, and 22% teenage-adult onset) were triaged into PCR-based RNA testing, with comparative RNA sequencing for 19 cases. Results: Informative RNA assay data were obtained for 96% of cases, enabling variant reclassification for 75% variants that can be used for genetic counseling (71%), to inform clinical care (32%) and prenatal counseling (41%). Variant-associated mis-splicing was highly reproducible for 28 cases with samples from ≥2 affected individuals or heterozygotes and 10 cases with ≥2 biospecimens. PCR amplicons encompassing another segregated heterozygous variant was vital for clinical interpretation of 22 of 79 variants to phase RNA splicing events and discern complete from partial mis-splicing. Conclusion: RNA diagnostics enabled provision of a genetic diagnosis for 64% of recruited cases. PCR-based RNA diagnostics has capacity to analyze 81.3% of clinically significant genes, with long amplicons providing an advantage over RNA sequencing to phase RNA splicing events. The Australasian Consortium for RNA Diagnostics (SpliceACORD) provide clinically-endorsed, standardized protocols and recommendations for interpreting RNA assay data.