Protocol for bulk RNA sequencing of enriched human neutrophils from whole blood and estimation of sample purity
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...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-03-01
|
| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166723000837 |
| _version_ | 1797895560638758912 |
|---|---|
| author | Anna L.K. Gonye Thomas J. LaSalle Samuel S. Freeman Miguel Reyes Nir Hacohen Alexandra-Chloé Villani Moshe Sade-Feldman |
| author_facet | Anna L.K. Gonye Thomas J. LaSalle Samuel S. Freeman Miguel Reyes Nir Hacohen Alexandra-Chloé Villani Moshe Sade-Feldman |
| author_sort | Anna L.K. Gonye |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-10T07:27:46Z |
| format | Article |
| id | doaj.art-9f70286ed7c742c8aa1618af8f718569 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-04-10T07:27:46Z |
| publishDate | 2023-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-9f70286ed7c742c8aa1618af8f7185692023-02-24T04:31:41ZengElsevierSTAR Protocols2666-16672023-03-0141102125Protocol for bulk RNA sequencing of enriched human neutrophils from whole blood and estimation of sample purityAnna L.K. Gonye0Thomas J. LaSalle1Samuel S. Freeman2Miguel Reyes3Nir Hacohen4Alexandra-Chloé Villani5Moshe Sade-Feldman6Center 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 authorCenter 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, USACenter for Cancer Research, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USABroad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USACenter for Cancer Research, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USACenter 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, USACenter 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 authorSummary: 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.http://www.sciencedirect.com/science/article/pii/S2666166723000837BioinformaticsSequence AnalysisCell BiologyCell IsolationSingle CellRNAseq |
| spellingShingle | Anna L.K. Gonye Thomas J. LaSalle Samuel S. Freeman Miguel Reyes Nir Hacohen Alexandra-Chloé Villani Moshe Sade-Feldman Protocol for bulk RNA sequencing of enriched human neutrophils from whole blood and estimation of sample purity STAR Protocols Bioinformatics Sequence Analysis Cell Biology Cell Isolation Single Cell RNAseq |
| title | Protocol for bulk RNA sequencing of enriched human neutrophils from whole blood and estimation of sample purity |
| title_full | Protocol for bulk RNA sequencing of enriched human neutrophils from whole blood and estimation of sample purity |
| title_fullStr | Protocol for bulk RNA sequencing of enriched human neutrophils from whole blood and estimation of sample purity |
| title_full_unstemmed | Protocol for bulk RNA sequencing of enriched human neutrophils from whole blood and estimation of sample purity |
| title_short | Protocol for bulk RNA sequencing of enriched human neutrophils from whole blood and estimation of sample purity |
| title_sort | protocol for bulk rna sequencing of enriched human neutrophils from whole blood and estimation of sample purity |
| topic | Bioinformatics Sequence Analysis Cell Biology Cell Isolation Single Cell RNAseq |
| url | http://www.sciencedirect.com/science/article/pii/S2666166723000837 |
| work_keys_str_mv | AT annalkgonye protocolforbulkrnasequencingofenrichedhumanneutrophilsfromwholebloodandestimationofsamplepurity AT thomasjlasalle protocolforbulkrnasequencingofenrichedhumanneutrophilsfromwholebloodandestimationofsamplepurity AT samuelsfreeman protocolforbulkrnasequencingofenrichedhumanneutrophilsfromwholebloodandestimationofsamplepurity AT miguelreyes protocolforbulkrnasequencingofenrichedhumanneutrophilsfromwholebloodandestimationofsamplepurity AT nirhacohen protocolforbulkrnasequencingofenrichedhumanneutrophilsfromwholebloodandestimationofsamplepurity AT alexandrachloevillani protocolforbulkrnasequencingofenrichedhumanneutrophilsfromwholebloodandestimationofsamplepurity AT moshesadefeldman protocolforbulkrnasequencingofenrichedhumanneutrophilsfromwholebloodandestimationofsamplepurity |