Single-nucleus RNA-seq of archived PAXgene whole blood samples

Description

Summary - No method has been developed for single-cell analysis of the large repositories of preserved whole blood samples stored in PAXgene Blood RNA tubes. To address this gap, two nuclei isolation techniques for single-nucleus RNA sequencing were evaluated: mechanical separation (MS), using an Acrodisc filter, and cell lysis (CL). While both methods captured nuclei from all major immune cell types, CL resulted in up to two orders of magnitude higher nuclei yields and less biased proportions of immune cells than MS. High ambient globin gene counts following CL were substantially reduced by CRISPR-guided globin gene depletion of complementary DNA, resulting in more sensitive and efficient gene detection per cell. Despite capturing only nuclear transcripts, CL-derived samples maintained similar cell-type proportions and gene expression as matched peripheral blood mononuclear cell samples, while retaining granulocytes. The CL isolation with globin depletion method enables comprehensive analysis of PAXgene whole blood samples at single-cell resolution. Experimental design - Three healthy donor blood was used to extract PBMC and prepare PAXgene blood RNA tubes. Frozen PAXgene tubes were later slowly thawed to perform MS and CL methods. MS focused on separating white blood cells by Acrodisc filter whereas CL focused on lysing of the blood pellet from PAXgene blood tubes. Matched PBMCs were also processed. All using 10x genomics v3.1 3’ gene expression assay. Extract protocol - Human donor blood A, B and C were commercially obtained from research blood components (https://www.researchbloodcomponents.com/). PBMCs were extracted and additional blood was stored in PAXgene blood RNA tubes. PBMCs were stored for long term in liquid nitrogen. PAXgene blood RNA tubes were stored at room temperature for 2 hours and then stored in minus 80⁰C. PBMCs were processed for scRNA-seq and PAXgene blood RNA tubes for snRNA-seq using 10X genomics v3.1 3' gene expression assay for single cell sequencing. Single cell suspension from PBMC was prepared after slowing thawing frozen PBMCs followed by washing in RPMI 0.1% BSA. Nuclei suspension was prepared from PAXgene blood RNA tubes for mechanical separation and Cell lysis as detailed in supplementary of preprint https://doi.org/10.1101/2025.03.06.641241 and in depth Cell lysis with depletion in protocols.io link. https://dx.doi.org/10.17504/protocols.io.n92ld6yxxg5b/v1. 10X genomics library construction protocol CG000315 Rev E was used for preparation of all libraries. CRIPSR Globin depletion-protcols.io https://dx.doi.org/10.17504/protocols.io.n92ld6yxxg5b/v1 Data processing step - The cellranger mkfastq pipeline (version 6.1.1; 10x Genomics) was used to demultiplex the base call files (BCLs) to FASTQ files, and then the cellranger count pipeline was used for downstream processing steps, including alignment to reference genome GRCh38, counting of unique molecular identifiers (UMIs), and cell calling. SRA - PRJNA1379454

Institutions

• AstraZeneca PLC

  Cambridgeshire, Cambridge

Categories

Funders

• AstraZeneca (United States)

  United States

Licence