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Single-cell RNA sequencing (scRNA-seq) has revolutionized the study of the cellular landscape of organs. Most single-cell protocols require fresh material, which limits sample size per experiment, and consequently, introduces batch effects. This is especially true for samples acquired through complex medical procedures, such as intestinal mucosal biopsies. Moreover, the tissue dissociation procedure required for obtaining single cells is a major source of noise; different dissociation procedures applied to different compartments of the tissue induce artificial gene expression differences between cell subsets. To overcome these challenges, we have developed a one-step dissociation protocol and demonstrated its use on cryopreserved gut mucosal biopsies. Using flow cytometry and scRNA-seq analysis, we compared this one-step dissociation protocol with the current gold standard, two-step collagenase digestion, and an adaptation of a recently published alternative, three-step cold-active Bacillus licheniformus protease digestion. Both cell viability and cell type composition were comparable between the one-step and two-step collagenase dissociation, with the former being more time-efficient. The cold protease digestion resulted in equal cell viability, but better preserves the epithelial cell types. Consequently, to analyze the rarer cell types, such as glial cells, larger total biopsy cell numbers are required as input material. The multi-step protocols affected cell types spanning multiple compartments differently. In summary, we show that cryopreserved gut mucosal biopsies can be used to overcome the logistical challenges and batch effects in large scRNA-seq studies. Furthermore, we demonstrate that using cryopreserved biopsies digested using a one-step collagenase protocol enables large-scale scRNA-seq, FACS, organoid generation and intraepithelial lymphocyte expansion. [ABSTRACT FROM AUTHOR]

Single-cell RNA sequencing (scRNA-seq) has revolutionized the study of the cellular landscape of organs. Most single-cell protocols require fresh material, which limits sample size per experiment, and consequently, introduces batch effects. This is especially true for samples acquired through complex medical procedures, such as intestinal mucosal biopsies. Moreover, the tissue dissociation procedure required for obtaining single cells is a major source of noise; different dissociation procedures applied to different compartments of the tissue induce artificial gene expression differences between cell subsets. To overcome these challenges, we have developed a one-step dissociation protocol and demonstrated its use on cryopreserved gut mucosal biopsies. Using flow cytometry and scRNA-seq analysis, we compared this one-step dissociation protocol with the current gold standard, two-step collagenase digestion, and an adaptation of a recently published alternative, three-step cold-active Bacillus licheniformus protease digestion. Both cell viability and cell type composition were comparable between the one-step and two-step collagenase dissociation, with the former being more time-efficient. The cold protease digestion resulted in equal cell viability, but better preserves the epithelial cell types. Consequently, to analyze the rarer cell types, such as glial cells, larger total biopsy cell numbers are required as input material. The multi-step protocols affected cell types spanning multiple compartments differently. In summary, we show that cryopreserved gut mucosal biopsies can be used to overcome the logistical challenges and batch effects in large scRNA-seq studies. Furthermore, we demonstrate that using cryopreserved biopsies digested using a one-step collagenase protocol enables large-scale scRNA-seq, FACS, organoid generation and intraepithelial lymphocyte expansion. [ABSTRACT FROM AUTHOR]

Celiac disease is an auto-immune disease in which an immune response to dietary gluten leads to inflammation and subsequent atrophy of small intestinal villi, causing severe bowel discomfort and malabsorption of nutrients. The major instigating factor for the immune response in celiac disease is the activation of gluten-specific CD4+ T cells expressing T cell receptors that recognize gluten peptides presented in the context of HLA-DQ2 and DQ8. Here we provide an in-depth characterization of 28 gluten-specific T cell clones. We assess their transcriptional and epigenetic response to T cell receptor stimulation and link this to genetic factors associated with celiac disease. Gluten-specific T cells have a distinct transcriptional profile that mostly resembles that of Th1 cells but also express cytokines characteristic of other types of T-helper cells. This transcriptional response appears not to be regulated by changes in chromatin state, but rather by early upregulation of transcription factors and non-coding RNAs that likely orchestrate the subsequent activation of genes that play a role in immune pathways. Finally, integration of chromatin and transcription factor binding profiles suggest that genes activated by T cell receptor stimulation of gluten‑specific T cells may be impacted by genetic variation at several genetic loci associated with celiac disease. [ABSTRACT FROM AUTHOR]