Protocol for bulk RNA sequencing of enriched human neutrophils from whole blood and estimation of sample purity

• Published in: STAR protocols Vol. 4; no. 1; p. 102125
• Main Authors: Gonye, Anna L.K., LaSalle, Thomas J., Freeman, Samuel S., Reyes, Miguel, Hacohen, Nir, Villani, Alexandra-Chloé, Sade-Feldman, Moshe
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17.03.2023; The Author(s); Elsevier
• Subjects: Bioinformatics; Cell Biology; Cell Isolation; Immunology; Molecular Biology; Protocol; RNAseq; Sequence Analysis; Single Cell; Immunology; Molecular Biology; Single Cell; Sequence Analysis; Bioinformatics; RNAseq; Cell Biology; Cell Isolation
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102125

Although neutrophils are the most abundant leukocyte in healthy individuals and impact outcomes of diseases ranging from sepsis to cancer, they remain understudied due to technical constraints of isolation, preservation, and sequencing. We present a modified Smart-Seq2 protocol for bulk RNA sequencing of neutrophils enriched from whole blood. We describe steps for neutrophil isolation, cDNA generation, library preparation, and sample purity estimation via a bioinformatic approach. Our approach permits the collection of large cohorts and enables detection of neutrophil transcriptomic subtypes. For complete details on the use and execution of this protocol, please refer to LaSalle et al. (2022)1 and Boribong et al. (2022).2 [Display omitted] •Streamlined negative selection enrichment of neutrophils from whole blood•Bulk purification of neutrophil RNA and high-quality, optimized library preparation•Quality control filtration and cell type abundance estimation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Although neutrophils are the most abundant leukocyte in healthy individuals and impact outcomes of diseases ranging from sepsis to cancer, they remain understudied due to technical constraints of isolation, preservation, and sequencing. We present a modified Smart-Seq2 protocol for bulk RNA sequencing of neutrophils enriched from whole blood. We describe steps for neutrophil isolation, cDNA generation, library preparation, and sample purity estimation via a bioinformatic approach. Our approach permits the collection of large cohorts and enables detection of neutrophil transcriptomic subtypes.