Optimization of single-nucleus RNA sequencing for superior characterization of the airway wall cellular landscape in pulmonary disease

Single-nucleus RNA sequencing (snRNA-seq) on biobanked specimens holds the promise to quickly reveal novel insights into the cellular complexity of lung tissues, particularly in chronic airway diseases like COPD and asthma. We optimized snRNA-seq protocols for airway wall tissue samples stored in OCT compound, focusing on key factors such as storage conditions, isolation procedure for nuclei, and multiplexing of samples with hashtag oligo (HTO) labelling against nucleopore proteins (Figure 1). The optimized method was successfully applied to a large number (30) of biobanked bronchial biopsies from ex-smokers with COPD, severe-early onset COPD and ex-smoking controls, as well as severe asthma mucosal cryo-biopsies collected as part of the 3TR Asthma Biologics Cohorte study at a secondary medical centre. The HTO staining was evaluated using the ‘HTO demux’ and ‘SoupOrCell’ packages and quality control and annotation pipelines were developed for this type of snRNA-seq data. The resulting dataset captures a broad spectrum of cell types from the airway wall across airway diseases and severities. This provides a valuable resource for studying the immune landscape and cellular interactions in disease progression. We found differences in cell-type composition between the clinical groups. This developed protocol will allow for large-scale clinical studies on lung disease. <fig><object-id>erjor;11/suppl_15/TP119/F1</object-id><object-id>F1</object-id><object-id>F1</object-id><graphic></graphic></fig>