Optimized Method for High-Quality Isolation of Single-Nuclei From Mosquito Fat Body for RNA Sequencing

Materials and reagents

Biological materials

1. Anopheles gambiae (G3, CDC strain) dissected body walls (25 tissues) from adult females

Reagents

1. Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: C1016-500G)

Caution: Irritant to the eyes.

2. 2 M potassium chloride (KCl) (Thermo Fisher Scientific, catalog number: AM9640G, 100 mL)

3. 1 M HEPES, pH 7.4 (Teknova, catalog number: H1030, 1,000 mL)

4. Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M8266-100G)

5. 5 M sodium chloride (NaCl) (KD Medical, catalog number: RGF-3270, 1,000 mL)

6. Sodium bicarbonate (NAHCO₃) (Sigma-Aldrich, catalog number: S6297-250G)

7. Sodium phosphate monobasic (NaH₂PO₄) (Sigma-Aldrich, catalog number: S3139-250G)

8. Sucrose (Sigma-Aldrich, catalog number: S0389-500G)

9. D-(+)-Trehalose dihydrate (Sigma-Aldrich, catalog number: T0167-25G)

10. Methanol (Thermo Fisher Scientific, catalog number: A412P-4, 4 L)

Caution: Flammable liquid and vapor. Toxic by inhalation, in contact with skin, and if swallowed. Handle inside of a safety cabinet.

11. Nuclease-free water (NFW), no DEPC (Thermo Fisher Scientific, catalog number: AM9938)

12. Nuclei Isolation kit, Nuclei PURE Prep (Sigma-Aldrich, catalog number: NUC201-1KT)

13. Dithiothreitol (DTT) (Thermo Fisher Scientific, catalog number: D1532, 1 g)

Caution: Irritant to skin and eyes. Harmful if swallowed.

14. Phosphate buffer saline (PBS) 10× (90 g NaCl, 1.44 g KH₂PO₄, 7.95 g NaHPO₄ in 1 L of water), pH 7.4 (KD-Medical, catalog number: RGF-3210)

15. RNASE inhibitor, protector RNASE inhibitor 10,000 units (Sigma-Aldrich, catalog number: 3335402001)

16. Ultrapure BSA 50 mg/mL (Thermo Fisher Scientific, catalog number: AM2618)

17. RNase Away (Molecular Bioproducts, catalog number: 7000)

18. Trypan blue stain, 0.4% (Gibco, catalog number: 15250061, 100 mL)

19. Triton X-100, molecular biology (Millipore, catalog number: 648466, 50 mL)

Solutions

1. Hemolymph-like saline (HLS buffer) (see Recipes)

2. 80% methanol solution (see Recipes)

3. Lysis buffer (see Recipes)

4. 1.8 M sucrose cushion solution (see Recipes)

5. Washing buffer 1× (see Recipes)

6. Resuspension buffer 1× (see Recipes)

Recipes

Note: Keep all solutions on ice at all times to maintain RNA integrity during the procedure. Prepare solutions fresh before nucleus isolation. Storage is not recommended, except for the HLS buffer.

1. HLS buffer

Notes:

1. Adjust the pH to 7.5, filter, and store aliquots at 4 °C.

2. Use solutions of CaCl₂ and MgCl₂ instead of powders to avoid precipitation in the saline.

3. This buffer was previously proposed by Singleton and Woodruff [18] to mimic Drosophila hemolymph osmolarity.

2. 80% methanol solution

Notes:

1. Each sample requires the use of 1 mL of 80% methanol solution.

2. This solution, used on step C1, is important to remove excess lipids.

Caution: Prepare and handle this solution inside a safety cabinet.

3. Lysis buffer

Note: Each sample requires the use of 0.5 mL of lysis buffer.

4. 1.8 M sucrose cushion solution

Notes:

1. Each sample requires the use of 2.8 mL of 1.8 M sucrose cushion solution.

2. Mix with a pipette or vortex until the solution looks clear and homogeneous. This solution is viscous due to the high sucrose concentration.

5. Washing buffer 1×

Note: Each sample requires the use of 2 mL of washing buffer.

6. Resuspension buffer 1×

Laboratory supplies

1. 1.5 mL microtubes (Axygen, catalog number: MCT-150-C-S)

2. 10 μL micropipette tips (TipOne 10 μL, graduated filter tips) (USA Scientific, catalog number: 1180-3810)

3. 200 μL micropipette tips (TipOne 200 μL graduated filter tips) (USA Scientific, catalog number: 1180-8810)

4. 1,250 μL micropipette tips (Purepoint barrier tips FT1250) (Alkali Scientific, catalog number: FT1250)

5. C-Chip disposable hemocytometer (iNCYTO, catalog number: DHC-N01-5 NI, Neubauer Improved)

6. Laboratory pipettes (10, 20, 200, and 1,000 μL)

7. Transfer pipette, sterile, polyethylene (Millipore Sigma, catalog number: Z350818-500EA)

8. WHEATON Dounce tissue grinder (WHEATON, catalog number: 357538, 1 mL)

9. Pellet pestles (Fisher Scientific, catalog number: 12-141-363)

10. 40 μm filter (Pluriselect, catalog number: 43-10040-40)

11. 20 μm filter (Pluriselect, catalog number: 43-10020-40)

Equipment

1. Refrigerated centrifuge (Eppendorf, model: 5417 R)

2. Rotor (Eppendorf, catalog number: F45-30-11)

3. Transmitted light and epifluorescence microscopes

Software and datasets

1. Cell ranger (10X genomics, version 7.0.0)

2. Seurat R package (Satija Lab, version 4.2.1)

3. DecontX R package (Campbell Lab, version 1.6.0)

4. All data have been deposited to the SRA database under BioProject accession number PRJNA1288431 (https://dataview.ncbi.nlm.nih.gov/object/PRJNA1288431?reviewer=he84ttfsjqcmttfp3ccg035vi8)

Procedure

A. General material setup

1. Before starting the protocol:

a. Prepare the solutions and place them on ice.

Note: If you have multiple samples at a time, you can prepare a larger mix respecting the proportions above.

b. Clean Dounce homogenizers and place them on ice.

Note: RNase Away may be used to avoid RNase activity. Rinse thoroughly with nuclease-free water.

c. Pre-chill the centrifuge to 4 °C.

d. Prepare 1.5 mL microcentrifuge tubes with 200 μL of HLS buffer and place them on ice. Prepare one tube per sample.

e. Prepare microcentrifuge tubes with 800 μL of methanol and put them on ice. Prepare one tube per sample.

B. Body wall dissection and tissue dehydration

1. Anesthetize the mosquitoes on ice. Pick a mosquito using fine forceps and pull the last segment of the abdomen to remove the ovaries, gut, and Malpighian tubules. Keep the body wall, with the fat body and associated tissues attached to the cuticle (Figure 1).

Figure 1. Graphical representation of mosquito body wall dissection

2. Dissect 25 body walls in HLS buffer and place them directly into 200 μL of HLS buffer in a single 1.5 mL microcentrifuge tube placed on ice.

Note: The optimal input consists of a pool of 25 body wall tissues per sample, yielding approximately 1 × 10⁶ nuclei.

3. Gently press the body wall tissue against the microcentrifuge tube in HLS buffer with a plastic pestle 4 times (Figure 2A).

Critical: The HLS buffer will get turbid. Do not squeeze the tissue too much or too hard to avoid cuticle pieces in the next steps of the protocol.

Figure 2. Nuclei isolation. (A) Fat body tissue after a gentle squeeze with a plastic pestle. (B, C) Tissue homogenate after methanol wash and centrifugation. (D) Nuclei extraction with lysis buffer and tissue Dounce homogenizer. (E) Sample homogenate laid on top of the sucrose cushion. The white arrow indicates the separation between the sample and the sucrose cushion. (F) After centrifugation, debris is found on the top layer and on the lateral of the tube (top white arrow), and nuclei are on the pellet on the bottom of the tube (bottom white arrow). (G) Nuclei pellet after washing the sample. (H) Nuclei on resuspension buffer.

4. Transfer the tissue homogenate to a tube with 800 μL of ice-cold methanol. Do not transfer the cuticle. Mix gently by inversion 3–4 times.

Note: If possible, use wide-bore tips or cut the tip with clean scissors to prevent cell damage at this point. You may clean the scissors with RNASE Away.

5. Repeat steps B1–5 until the desired number of samples is acquired.

Note: Keep samples in 80% methanol/HLS on ice until all samples are collected.

C. Nuclei isolation

1. Lipid removal

a. Centrifuge at 500× g for 5 min at 4 °C. Remove the supernatant without disrupting the pellet.

b. Wash with 1 mL of ice-cold 80% methanol solution. Pipette gently to resuspend the pellet (Figure 2B).

Critical: The mosquito fat body has a large amount of lipids. This methanol wash fixes the tissue and significantly reduces the lipid content in the sample, which is crucial for downstream processing.

c. Centrifuge at 500× g for 5 min at 4 °C. Remove the supernatant without disrupting the pellet (Figure 2C).

2. Tissue dissociation

a. Add 500 μL of ice-cold lysis buffer and mix gently with a pipette. Pipette gently to resuspend the pellet.

Notes:

1. If possible, use a wide-bore pipette tip or cut the tip with clean scissors. You may clean the scissors with RNASE Away.

2. It is not recommended to keep the tissues for longer than 10 min in lysis buffer. Longer exposure to the lysis buffer may cause loss of nuclei quality.

b. Transfer the sample to the pre-chilled 1 mL Dounce homogenizer. Use the loose pestle to perform 15 gentle strokes. Repeat this step and perform 15 gentle strokes but with the tight pestle (~2–3 min). At this point, the sample should look turbid and with little to no foam (Figure 2D).

Notes:

1. Perform the strokes gently. Keep the pestle submerged the entire time to avoid foam or bubble formation.

2. Keep the sample on ice the entire time to avoid RNA degradation.

c. Add 900 μL of 1.8 M sucrose cushion solution and mix gently with a transfer pipette.

Critical: The 1.8 M sucrose cushion solution will dilute the lysis buffer, minimizing its action on the sample. It also adjusts the concentration of sucrose in the sample to be used as an overlay in the sucrose cushion step.

Note: If processing multiple samples, it is recommended to dilute the lysis buffer and remove it by centrifugation until you finish processing all the samples.

3. Sucrose cushion

Note: If processing blood-fed mosquitoes, split the sample into four microcentrifuge tubes with sucrose cushion. Blood-fed mosquitoes generate more cellular debris, and splitting the sample into more tubes improves the efficiency of the sucrose cushion cleanup.

a. Add 500 μL of 1.8 M sucrose cushion solution to a new microcentrifuge tube and centrifuge at 1,000× g for 30 s. Prepare two microcentrifuge tubes per sample. Keep it on ice.

b. Carefully overlay 700 μL of the sample homogenate on top of the sucrose cushion prepared in the previous step in each microcentrifuge tube (Figure 2E).

Critical: The sample should be gently applied along the tube wall without disrupting the sucrose cushion. A distinct layer should be visible above the sucrose cushion.

c. Without disturbing the layers, transfer the microcentrifuge tubes to a pre-chilled microcentrifuge. Centrifuge samples at 13,000× g for 45 min at 4 °C.

d. Carefully remove the tube from the centrifuge and place it on ice. The nuclei should be pelleted at the bottom of the tube. Cellular debris may appear on top of the supernatant, along the tube wall, and/or at the interface between the tissue homogenate and the sucrose cushion. Gently remove the entire supernatant, including both the homogenate layer and the sucrose cushion, without disturbing the pellet (Figure 2F).

Notes:

1. If using a fixed-angled rotor, beware of the debris on the wall of the microcentrifuge tube.

2. If possible, use a swinging bucket rotor to improve the efficiency of the sucrose cushion and yield of the final sample with less debris.

e. Wash the pellet twice with 1 mL of washing buffer. Centrifuge at 500× g for 5 min at 4 °C. Remove the supernatant without disrupting the pellet (Figure 2G).

Notes:

1. Increasing wash volumes and using a swing bucket rotor during centrifugation may improve the removal of leftover debris, lipids, and other contaminants.

2. Cell strainers of 20–40 μm may be used to remove large debris remaining on the sample after two washes. Attach the strainer to a microcentrifuge tube and filter the sample in washing buffer. Note that filtering can trap some nuclei and may reduce yield.

3. Increase BSA concentration in the resuspension buffer up to 2% if nuclei aggregates are >5%.

f. Resuspend the pellet with gentle taps on the microcentrifuge tubes. Add 100 μL of resuspension buffer to the first tube. Gently mix by pipetting and then transfer all contents of the first tube into the second tube to combine them. The final volume will be approximately 100 μL per sample (Figure 2H).

Note: Adjust the final resuspension volume according to the number of tissues used and nuclei yield. Do not use less than 50 μL to resuspend the sample, as per the recommendations of 10× protocols. Nuclei yield is optimal between 700 and 1,200 nuclei/μL.

4. Counting and visualizing the nucleus

a. Perform nucleus manual counting using the Neubauer improved chamber under the microscope. Use filtered trypan blue (0.1%) to observe and count the nuclei easily. Use light microscopy and Hoechst (1:10,000) staining to assess the quality of nuclei extraction.

Note: Good quality nuclei should have an intact or undamaged nuclei membrane, without blebs.

Data analysis

Cell lysis is necessary to extract nuclei for single-nuclei library preparation; however, it can introduce ambient RNA contamination into the samples. Although cell washing helps, it often fails to fully eliminate this contamination. Ambient RNA can negatively impact data quality by introducing noise, leading to poor-quality cells and cofounding gene expression patterns during clustering and integration analysis. To address this, computational methods like R packages DecontX [19] and SoupX [20] use Bayesian frameworks to estimate contamination across all cells. These tools can either filter out low-quality cells [19] or adjust their expression profiles to correct for contamination [20]. More recently, complementary approaches have emerged. For example, scCDC [21] identifies globally contaminating genes and performs decontamination by adjusting the expression counts of the specific genes within each cell, rather than modeling contamination at the cell level. These approaches, whether used alone or in combination, enhance the accuracy and interpretability of single-nucleus RNA-seq data by effectively mitigating the impact of ambient RNA.

In this study, we generated a single-nucleus gene matrix by aligning raw sequence reads from the Anopheles gambiae PEST (AgamP4.14, v59) reference genome using the Cell Ranger pipeline (version 7.0.0, 10X Genomics, Pleasanton, CA). Alignment and quantification were performed with the “cellranger count” function, using the parameters “--include-introns=true” and “--expect-cells=10000” to generate both raw and filtered gene-barcode matrices. Correction was performed using the decontX algorithm from the celda R package [19], which models and removes contamination from ambient background RNA in snRNA-seq data. Additional quality filtering was performed based on the number of features, with thresholds set at 200–1,500 for the sugar-fed sample and 200–2,000 features for the blood-fed sample. Cells were also filtered based on mitochondrial and ribosomal gene expression, as a proxy of ambient RNA contamination, with maximum allowable percentages set at 15% for mitochondrial genes and 15%–25% for ribosomal genes on sugar-fed and blood-fed samples, respectively.

Validation of protocol

The protocol described here was tested on sugar-fed and blood-fed mosquitoes and yielded reproducible results. Nuclei isolation consistently yielded optimal nuclei concentration for 10X genomics kits, ranging from 700 to 1,200 nuclei/μL. Nuclei quality was assessed using light and fluorescent microscopy with Hoechst staining. High-quality nuclei with varying sizes were observed, characterized by intact nuclear membranes (Figure 3A, B). High-quality nuclei are characterized by an intact nuclear membrane (Figure 3A’, B’), whereas poor-quality nuclei show membrane blebbing or disruption (Figure 3C, D). cDNA libraries were generated using the Chromium Next GEM Single Cell 3′ Reagent Kit v3.1. cDNA and library quality and quantification were evaluated using capillary electrophoresis with the Agilent TapeStation system (Figure 3E–H). Ambient RNA removal and filtering thresholds were applied to ensure that only high-quality cells were retained in the sugar-fed and blood-fed datasets (Table 1). Violin plots show the data distribution before and after quality cutoffs were applied (Figure 4A, B). Notably, these distributions differ between sugar-fed and blood-fed samples, likely reflecting the extensive transcriptional reprogramming that occurs in the mosquito fat body following a blood meal.

Figure 3. Isolated nuclei from Anopheles gambiae fat body. (A) Bright field and (B) nuclei staining (blue). Scale bar: 40 μm. (A’, B’) Zoom-in showing good-quality nuclei of different sizes. Scale bar: 7 μm. (C) Bright field showing poor-quality nuclei, with blebbing. Scale bar: 7 μm. (D) Bright field of the Newbauer chamber showing good (right) and poor (left) nuclei. Scale bar: 10 μm. (E, F) cDNA sample intensity normalized and acquired with TapeStation for sugar-fed (E) and blood-fed (F) samples. (E, F) Library sample intensity normalized and acquired with TapeStation for sugar-fed (G) and blood-fed (H) samples.

Table 1. Summary of parameters before and after quality control (QC) metrics were applied to the single-nucleus RNA sequencing data from sugar-fed and blood-fed dissociated tissues.

*Mean reads per cell after setting quality thresholds were estimated based on the total number of reads and the recovered number of nuclei after QC analysis.

**The number of recovered cells is higher than the number of loaded cells before QC. That is a common observation in single-nuclei RNA-seq that may be caused by ambient RNA contamination.

***Low fraction reads in nuclei indicate that many reads were not assigned to nuclei-associated barcodes, which could mean contamination with ambient RNA or cells with low RNA content.

Figure 4. Data distribution before and after quality filtering. (A, B) Distribution of the number of genes (nFeature), number of UMIs (nCount), percent of mitochondrial gene expression, and percentage of ribosome gene expression contamination score in sugar-fed and blood-fed samples, before (A) and after (B) quality filtering.

This protocol (or parts of it) has been used and validated in the following research article(s):

• de Carvalho et al. [22]. Cell-specific responses of Anopheles gambiae fat body to blood feeding and infection at a single nuclei resolution. bioRxiv 2025.07.29.667437; doi: https://doi.org/10.1101/2025.07.29.667437

General notes and troubleshooting

Libraries were constructed using the Chromium Single Cell 3′ Kit v3.1 (Dual Index) (10X Genomics) following the manufacturer’s standard protocol. In the Chromium workflow, gel bead-in-emulsion (GEM) formation is a critical initial step, and the quality of the nuclei suspension can influence emulsion integrity. Samples with excessive lipids or residual debris can interfere with stable GEM formation or the microfluidic capillary system. Biphasic or heterogeneous GEM formation may lead to library prep failure. For this reason, a clean, low-debris nuclei preparation is essential for reliable library generation.

The 10X Chromium protocol has a correlation table to adjust cDNA and library amplification cycles based on the measured DNA mass of each sample, ensuring appropriate amplification while minimizing technical artifacts during sequencing. In cases where libraries exhibit an over-representation of short fragments (<200 bp), the manufacturer recommends an additional cleanup step. Although this may reduce yield, it improves downstream sequencing performance by reducing the contribution of nonspecific low fragments.

Sequencing was performed on an Illumina NextSeq 2000 platform using paired-end 150 bp reads. The targeted sequencing depth was approximately 20,000 reads per nucleus, or until library saturation was achieved, consistent with standard recommendations for single-nucleus 3′ gene expression profiling.

Troubleshooting (Table 2)

Table 2. Summary of main issues and suggested solutions for fat body nuclei isolation.

Acknowledgments

S.S.D.C and C.B.-M. conceptualized and planned the study. S.S.D.C. performed test trials and troubleshooting. S.S.D.C. performed immunofluorescence and image analysis. S.S.D.C. and C.B.-M. wrote the manuscript. S.S.D.C., C.M., and C.B.-M. edited the manuscript. S.S.D.C. and C.M. performed data analysis. We thank Kevin Lee, Yonas Gebremicale, Andre Laughinghouse, and Claudio Meneses for insectary support. This work was supported by the Intramural Research Program of the Division of Intramural Research Z01AI000947, NIAID. The graphical overview was created in https://BioRender.com.

This research was supported by the Intramural Research Program of the National Institutes of Health (NIH). The contributions of the NIH author(s) were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. This protocol was used in de Carvalho et al. [22]. Cell-specific responses of Anopheles gambiae fat body to blood feeding and infection at a single nuclei resolution. bioRxiv 2025.07.29.667437; doi: https://doi.org/10.1101/2025.07.29.667437.

Competing interests

The authors declare no conflicts of interest.

Ethical considerations

Public Health Service Animal Welfare Assurance #A4149-01 guidelines were followed according to the National Institutes of Health (NIH) Office of Animal Care and Use (OACU). These studies were done according to the NIH animal study protocol (ASP) approved by the NIH Animal Care and User Committee (ACUC), with approval ID ASP-LMVR5.

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