从全血中富集的人中性粒细胞的批量 RNA 测序方案及样本纯度评估。
Protocol for bulk RNA sequencing of enriched human neutrophils from whole blood and estimation of sample purity.
机构信息
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.
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.
出版信息
STAR Protoc. 2023 Mar 17;4(1):102125. doi: 10.1016/j.xpro.2023.102125. Epub 2023 Feb 23.
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) and Boribong et al. (2022)..
虽然中性粒细胞是健康个体中最丰富的白细胞，并且对从脓毒症到癌症等各种疾病的结果有影响，但由于其分离、保存和测序的技术限制，它们的研究仍然不足。我们提出了一种改良的 Smart-Seq2 方案，用于从全血中富集的中性粒细胞进行批量 RNA 测序。我们描述了中性粒细胞分离、cDNA 生成、文库制备以及通过生物信息学方法估计样品纯度的步骤。我们的方法允许收集大量队列，并能够检测中性粒细胞转录组亚型。有关此方案的使用和执行的完整详细信息，请参阅 LaSalle 等人（2022 年）和 Boribong 等人（2022 年）。
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