The Simple prEservatioN of Single cElls method for cryopreservation enables the generation of single-cell immune profiles from whole blood.

Introduction: Current multistep methods utilized for preparing and cryopreserving single-cell suspensions from blood samples for single-cell RNA sequencing (scRNA-seq) are time-consuming, requiring trained personnel and special equipment, so limiting their clinical adoption. We developed a method, Simple prEservatioN of Single cElls (SENSE), for single-step cryopreservation of whole blood (WB) along with granulocyte depletion during single-cell assay, to generate high quality single-cell profiles (SCP). Methods: WB was cryopreserved using the SENSE method and peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved using the traditional density-gradient method (PBMC method) from the same blood sample (n=6). The SCPs obtained from both methods were processed using a similar pipeline and quality control parameters. Further, entropy calculation, differential gene expression, and cellular communication analysis were performed to compare cell types and subtypes from both methods. Results: Highly viable (86.3 ± 1.51%) single-cell suspensions (22,353 cells) were obtained from the six WB samples cryopreserved using the SENSE method. In-depth characterization of the scRNA-seq datasets from the samples processed with the SENSE method yielded high-quality profiles of lymphoid and myeloid cell types which were in concordance with the profiles obtained with classical multistep PBMC method processed samples. Additionally, the SENSE method cryopreserved samples exhibited significantly higher T-cell enrichment, enabling deeper characterization of T-cell subtypes. Overall, the SENSE and PBMC methods processed samples exhibited transcriptional, and cellular communication network level similarities across cell types with no batch effect except in myeloid lineage cells. Discussion: Comparative analysis of scRNA-seq datasets obtained with the two cryopreservation methods i.e., SENSE and PBMC methods, yielded similar cellular and molecular profiles, confirming the suitability of the former method's incorporation in clinics/labs for cryopreserving and obtaining high-quality single-cells for conducting critical translational research.