概要: The Drosophila lymph gland (LG) is the larval hematopoietic organ comprised of multipotent prohemocytes and mature hemocytes and has been a valuable model for understanding mechanisms underlying the hematopoiesis and immune responses. There are three types of mature hemocytes in the lymph gland; plasmatocytes, lamellocytes, and crystal cells, all of which are analogous to myeloid-lineage blood cells. To date, the Drosophila hematopoietic system has been primarily defined by classical genetic methods that may limit the promise of its advantage as a model. In this study, we used single-cell RNA sequencing method to comprehensively profile the heterogeneity of developing myeloid hemocytes in the wild-type lymph gland and their transitions upon active immunity caused by wasp infestation. Moreover, we compared hemocytes originated from the embryonic and the lymph gland hematopoiesis and unraveled genetic and cellular diversity of two different ancestries. Finally, hemocytes of the Drosophila lymph gland and human immune cells are contrasted at a transcriptome level, highlighting evolutionary conservations of myeloid cells across species. Overall, our single-cell RNA seq analyses disclose the development of myeloid hemocytes at a single-cell level and provide comparative insights into two different lineages of hemocytes in Drosophila and perspectives on the evolution of myeloid cells, serving as an ample resource for revealing essentials of the hematopoiesis.
项目整体设计: Single-cell RNA-sequencing of Drosophila larvae LGs or circulating hemocytes under normal or wasp infested states using Drop-seq
100 to 150 primary LGs were dissected and kept in 200 µl ice-cooled Schneider’s medium (Gibco, 21720024). After dissection, centrifugation at 3,000 rpm and 4 ℃ for 5 minutes was done. Supernatant was discarded and 300 µl of room-temperature Schneider’s medium was added to the lymph gland primary lobes. 300 µl of Papain (Worthington, LK003178) pre-heated to 37 ℃, and 4.1 µl of Liberase TM (Roche, 5401119001) were added and gently mixed. The samples were incubated for 20 minutes with gentle nutation. At 5-, 10-, and 15-minute time points of incubation, samples were mixed using 200p pipette. Enzymes were inactivated with 100 µl of ice-cooled Schneider’s medium, and samples were kept on ice. Suspended cells were passed through a 40 µm cell strainer (Corning, 352340). After, centrifugation at 3,000 rpm and 4 ℃ for 5 minutes was done. Supernatant was discarded, and 1X filtered sterile PBS was added to cells. Final concentration of cells was fixed to 300 cells/µl for a total of 600 µl.
建库方案:
All the Drop-seq and cDNA synthesis methods were followed by previous study (Macosko, Evan Z., et al. "Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets." Cell 161.5 (2015): 1202-1214.). The concentration of beads was fixed to 300 beads/u. Around 10 minutes Drop-seq run performed for each experiment. single-cell RNA-seq (Drop-seq)
测序信息
分子类型:
poly(A)+ RNA
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PAIRED
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Forward
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ILLUMINA
测序仪型号:
Illumina NextSeq 550
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Specific
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文章
Single-cell transcriptome maps of myeloid blood cell lineages in Drosophila.
