Research on sNuc-Seq of Drosophila Hemocyte

For my inaugural master's project, I focused on the optimization of single-nuclei RNA sequencing (sNuc-Seq) for the analysis of Drosophila hemocytes in both pupal and adult stages.

The study aimed to address several key objects:

1. Investigate the changes in hemocyte development during Drosophila metamorphosis at the transcriptome level, focusing on pupal hemocytes, as single-cell RNA sequencing had previously been performed on larval hemocytes.

2. Understand the factors and processes involved in the transition of hemocytes from a mobile to sessile state.

3. Explore the relationship between larval circulating hemocytes and hemocytes located in the lymph gland.

This research project aimed to fill a knowledge gap by providing insights into the molecular mechanisms underlying hemocyte development, metamorphosis-related changes, and the dynamics of different hemocyte populations within Drosophila, with a particular emphasis on pupal hemocytes.

First, I identified the optimal time point during the metamorphosis from larva to pupa for sNuc-Seq, as illustrated in the figure.

Following the protocol developed by Dr. Hongjie Li for nuclei isolation from cells, I encountered success in this step.

However, I encountered challenges during the process of dissecting pupae and obtaining pure hemocytes. Consequently, I opted to utilize Fluorescence-Activated Cell Sorting (FACS) to collect hemocytes and assess the expression of hemocyte markers in pupae. To facilitate the sorting of hemocytes, I initially examined the expression of srp and Hml. Subsequently, I generated Drosophila strains, including srp-Gal4 x UAS-lam-GFP and HmlΔ-Gal4 x UAS-lam-GFP. Ultimately, I created strains that enabled the tracking of hemocytes based on both srp and Hml expression; srp-Gal4;HmlΔ-Gal4 x UAS-lam-GFP and srp;UAS-lam-GFP x HmlΔ-Gal4.

Using the strains that I generated, I conducted dissection-hemocyte sorting-nuclei isolation procedure several times to refine the protocol. This iterative process proved successful, as you can see in the figure.

One of the primary objectives of performing sNuc-Seq on Drosophila pupae and adults was to decipher the transcriptomic changes in hemocytes during metamorphosis from larva to adult. To facilitate the tracking of this metamorphic process, I generated a strain, hand>GFP x UAS-GTRACE and handΔ-Gal4 x UAS-GTRACE. These strains allowed me to monitor and trace the transformation of larval to adult hemocytes. I initially assessed the expression patterns in larval hemocytes to lay the groundwork for this study.

My research project involved the creation of Drosophila strains for sNuc-Seq on hemocytes, the optimization of sorting Drosophila pupae and adults using FACS, and isolation of nuclei according to the protocol developed by Dr. Hongjie Li. During the course of my work, I had the opportunity to run Drop-seq several times while fine-tuning the entire process.