Cell lysis plus depletion single-nucleus RNA-seq (CL-D snRNAseq) v1

PAXgene Blood RNA Tubes (Qiagen) are commonly used for the direct collection of whole blood from donors. These tubes contain a proprietary solution that stabilizes RNA in blood cells, allowing for long-term storage at–80°C. For routine preparation of frozen PAXgene blood specimens for single-nucleus RNA sequencing (snRNA-seq), it has been demonstrated that nuclei must be isolated and RNA from these nuclei must be profiled using a specific, reproducible protocol. Here, we present a protocol focused on the isolation of nuclei from frozen PAXgene blood specimens, followed by library preparation using the 10x Genomics Single Cell Gene Expression Solution. Additionally, we describe the use of CRISPR-based digestion to remove cDNA generated from globin gene RNA, optimizing the quality of sequencing data.