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

Anna L K Gonye; Thomas J LaSalle; Samuel S Freeman; Miguel Reyes; Nir Hacohen; Alexandra-Chloé Villani; Moshe Sade-Feldman

doi:10.1016/j.xpro.2023.102125

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

STAR Protoc. 2023 Mar 17;4(1):102125. doi: 10.1016/j.xpro.2023.102125. Epub 2023 Feb 23.

Authors

Anna L K Gonye¹, Thomas J LaSalle², Samuel S Freeman³, Miguel Reyes⁴, Nir Hacohen³, Alexandra-Chloé Villani⁵, Moshe Sade-Feldman⁶

Affiliations

¹ Center for Cancer Research, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Cellular and Molecular Medicine Graduate Program, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Electronic address: agonye1@jhmi.edu.
² Center for Cancer Research, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Program in Health Sciences and Technology, Harvard Medical School & Massachusetts Institute of Technology, Boston, MA, USA.
³ Center for Cancer Research, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁴ Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁵ Center for Cancer Research, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Center for Immunology and Inflammatory Diseases, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA; Department of Medicine, Harvard Medical School, Boston, MA, USA.
⁶ Center for Cancer Research, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Medicine, Harvard Medical School, Boston, MA, USA. Electronic address: msade-feldman@mgh.harvard.edu.

Abstract

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.

Keywords: Bioinformatics; Cell Biology; Cell Isolation; Immunology; Molecular Biology; RNAseq; Sequence Analysis; Single Cell.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Base Sequence
Humans
Leukocytes
Neutrophils*
Sepsis*
Sequence Analysis, RNA

Abstract

Publication types

MeSH terms

Grants and funding