Comparison of droplet- vs. microwell-based single-cell RNA sequencing methods for the analysis of human bronchoalveolar lavage cells after cryopreservation.

Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool to investigate tissue biology and heterogeneity by providing gene expression data at the single cell level. Yet, the need to use fresh material has hampered investigations of human samples, due to obvious logistic reasons. In this project, we aimed to implement and compare cryopreservation methods on human bronchoalveolar lavage (BAL) cells in combination with two scRNA-seq methods, namely droplet-based (10x Chromium™), and microwell-based (Honeycomb™) entrapment.To this end, we aimed to analyze 4 BAL samples obtained from routine diagnostic procedures at the University Hospital (CHU, ULiège, Belgium). The BAL cells were divided into 2 fractions and processed using both techniques according to the manufacturers’ instructions and advices.Our results indicate that the droplet-based method showed a higher recovery rate of intact cells after sequencing and quality control filtering as compared to the microwell-based method. Both methods were able to capture ARN transcripts from fragile granulocytic cells. The proportion of the identified cells types, however, was different between the two methods. Of note, the average number of transcripts per cell was substantially higher with the droplet-based method, while the percentage of detected mitochondrial genes, indicative of stressed or dying cells, was much lower with the droplet-based method compared to the microwell-based method. So far, our results indicate that, in our hands and for the analysis of cryopreserved BAL cells, the droplet-based method display several advantages over the microwell-based method. Our data highlight the importance of determining the most appropriate methodology to obtain high-quality scRNA-seq data before embarking on expensive high-troughput analyses.