Parallel recovery of chromatin accessibility and gene expression dynamics from frozen human Regulatory T cells
Authors: Wong Ying Y. , Harbison Jessica E. , Hope Christopher M. , Gundsambuu Batjargal , Brown Katherine A. , Wong Soon W. , Brown Cheryl , Couper Jennifer J. , Breen Jimmy , Liu Ning , Pederson Stephen M. , Köhne Maren , Klee Kathrin , Schultze Joachim , Beyer Marc , Sadlon Timothy , Barry Simon C. 
Published: August 2022

Abstract
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.