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

STAR Protocols (Mar 2023)

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

Affiliations

Anna L.K. Gonye: 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; Corresponding author
Thomas J. LaSalle: 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
Samuel S. Freeman: Center for Cancer Research, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA
Miguel Reyes: Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
Nir Hacohen: Center for Cancer Research, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA
Alexandra-Chloé Villani: 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
Moshe Sade-Feldman: 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; Corresponding author

Journal volume & issue: Vol. 4, no. 1
p. 102125

Abstract

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Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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