A robust method to isolate Drosophila fat body nuclei for transcriptomic analysis

ABSTRACT Gene expression profiles are typically described at the level of the tissue or, often in Drosophila, at the level of the whole organism. Collapsing the gene expression of entire tissues into single measures averages over potentially important heterogeneity among the cells that make up that tissue. The advent of single-cell RNA-sequencing technology (sc-RNAseq) allows transcriptomic evaluation of the individual cells that make up a tissue. However, sc-RNAseq requires a high-quality suspension of viable cells or nuclei, and cell dissociation methods that yield healthy cells and nuclei are still lacking for many important tissues. The insect fat body is a polyfunctional tissue responsible for diverse physiological processes and therefore is an important target for sc-RNAseq. The Drosophila adult fat body consists of fragile cells that are difficult to dissociate while maintaining cell viability. As an alternative, we developed a method to isolate single fat body nuclei for RNA-seq. Our isolation method is largely free of mitochondrial contamination and yields higher capture of transcripts per nucleus compared to other nuclei preparation methods. Our method works well for single-cell nuclei sequencing and can potentially be implemented for bulk RNA-seq.


Introduction
The insect fat body is a highly multifunctional tissue that regulates diverse physiological processes, such as nutrient storage and metabolic control, immune responses to infection, and production of proteins essential for egg provisioning [1]. This single tissue thus shares function with several vertebrate tissues, including liver and adipose tissue. The fat body is an extremely dynamic tissue that exhibits dramatic expression changes in response to physiological stimulus [2]. It, therefore, is an important tissue to understand. The diverse functions of the fat body imply that there may be cellular heterogeneity within the tissue, and spatially restricted morphological and functional heterogeneity have previously been observed [3,4]. Single-cell RNAseq (sc-RNAseq) is a technique that enables transcriptomic profiling of individual cells [5], which could be invaluable for studying the fat body. However, the success of sc-RNAseq relies on clean, gentle, and rapid dissection of the tissue of interest and dissociation of individual cells. The adult fat body of Drosophila melanogaster is a large and fragile tissue that is distributed throughout the body [1] and is, therefore, more difficult than other tissues to analyse at a single-cell level. In this manuscript, we compare four methods for isolating fat body cells and nuclei prior to sc-RNAseq. In our hands, isolation of intact fat body cells is infeasibly challenging and results in unacceptably high cellular mortality (see Supplement). Standard protocols to isolate individual nuclei using a sucrose gradient or low-speed centrifugation were successful in capturing nuclei but carried unacceptably high levels of contamination with mitochondria (see Supplement). We ultimately developed a modified method that combines careful tissue dissection and fixation, cell lysis, and nuclear isolation over a sucrose density gradient to generate high-yield, high-purity nuclear isolations that are suitable for transcriptomic profiling. Our method resulted in high-quality RNA that was suitable for single-cell transcriptomic profiling by RNA sequencing [6].

Results
Enzymatic dissociation is the most common method to dissociate a tissue into a single cell suspension. We noticed that the Drosophila fat body cells are fragile and rapidly die when subjected to enzymatic dissociation (described in Supplement). Therefore, this was not a suitable approach from our perspective. Studies [7][8][9] have shown that transcriptomic profiles correlate strongly between nuclei and cells, meaning that nascent transcripts in the nucleus are broadly representative of the standing mRNA pool in the cell and suggesting they could be used for transcriptomic profiling. We tested various methods to prepare nuclei suspension from the fat body tissue. We tested two methods, sucrose cushion gradient and low-speed centrifugation (described in Supplement), which resulted in an unacceptably high proportion of mitochondrial reads ( Figure 1) in the sequencing data. Our objective was to generate high-quality nuclear gene expression data with low contamination from mitochondria and other cellular debris, so we optimized a protocol to isolate individual intact nuclei.
Using our optimized protocol, we saw a dramatic reduction in mitochondrial contamination. Fewer than 5% of the purified nuclei were associated with 20% or higher mitochondrial reads (Figure 1), and there was a considerable improvement in the number of reads and distinct genes obtained per nucleus using the optimized protocol ( Table 1). As a part of routine sample quality control for single-cell data analysis, we only consider nuclei expressing 20 genes or more when considering genes that are expressed in at least five cells. Upon analysing the raw dataset with Seurat v3.1 [10], our optimized protocol showed a significantly higher number of genes captured per nucleus ( Figure 2). The dramatic reduction in the number of mitochondrial reads and a larger number of unique expressed genes detected per nucleus in our optimized protocol represents a major improvement over the other two protocols tested. Our method was robust and gave a similar value for low mitochondrial read contamination and a high number of genes captured when applied to eight fat body samples subjected to different biological treatments [6]. The optimized protocol we developed is as follows:

Adult haemolymph-like saline (HLS)
For dissection and storing tissues, haemolymph-like saline of osmolarity suitable for adult flies [11] was

Protocol
(1) Autoclave micro-centrifuge tubes (Eppendorf# 022431048), pipette tips, Figure 2. Box-overlaid-violin plot showing the number of unique expressed genes detected per nucleus after isolation with three different nuclei preparation protocols. The expressed gene counts were generated from the single-nucleus sequence data analysed using Seurat [10]. Our optimized protocol yielded a higher number of unique expressed genes detected per nucleus.
(2) Keep micro-centrifuge tubes and Dounce homogenizers at 4°C so that everything is chilled before use. We further removed cellular debris using sucrose gradient centrifugation. Using Sigma kit (NUC201-KT), we followed the protocol using instructions provided by 10X Chromium [12]. The protocol is as follows:

Discussion
Here we provide an optimized method for preparing a nuclear suspension suitable for transcriptomics. Our method uses a combination of detergents and gentle centrifugations over a sucrose gradient to generate a purified nuclear suspension with low mitochondrial contamination and a higher number of genes captured per nucleus. Fixing the samples immediately upon dissection preserves the quality of the RNA and results in excellent transcriptomic data [6]. Detection of a greater number of expressed genes per cell allows researchers to make more accurate predictions about the biology of single cells. The ability to robustly capture more genes per cell has driven advancement in single-cell transcriptomics protocols, such as Smart-seq protocols [13] or different chemistries of 10x genomics 3 sequencing kits [14]. Single-cell sequencing technology requires dissociation protocols that can provide a high yield of intact living cells from which a standing mRNA pool can be reliably recovered. However, cell dissociation protocols are delicate, and the precise protocol needs to be specifically optimized for the tissue type of interest. This may prove challenging for fragile tissues, such as the insect adult fat body, whose cells are unstable and rapidly die upon dissociation. On the contrary, nuclei can be isolated from any cell or tissue type. Since the nascent transcript pool in the nucleus is strongly correlated with the standing mRNA pool in the cytoplasm [7][8][9], sequencing nuclei can provide a reliable alternate strategy for sequencing any tissue at single-cell resolution.
Although our protocol was specifically developed for Drosophila melanogaster, we expect that it can be broadly applied. The fat body is a major tissue in all insects and performs several key functions including immune response, metabolism, production of egg yolk and vitellogenins, and xenobiotic detoxification. Single-cell/nucleus sequencing provides a tremendous opportunity to study this crucial insect tissue and understand the cellular basis for functional diversity within the tissue. We expect that the protocol we present here can be readily applied to the fat body of non-Drosophila insects and can be adapted for other tissue types as well.