
==== Front
STAR Protoc
STAR Protoc
STAR Protocols
2666-1667
Elsevier

S2666-1667(24)00463-5
10.1016/j.xpro.2024.103298
103298
Protocol
Protocol for optimized nasal mucosa sample processing to obtain high-quality scRNA-seq and scATAC-seq data
Huang Yaling 145
Wu Yisha 1234
Han Shikai 1234
Wang Qiaoling 14
Cong Guomingxiu 1
Liu Zhongzhen 1
Guan Shuyan 1
Huang Xiaojuan 1
Liu Ying 1
Yin Jianhua yinjianhua@genomics.cn
13∗
Xue Jinmei xjment@126.com
23∗∗
Liu Chuanyu liuchuanyu@genomics.cn
136∗∗∗
1 BGI Research, Shenzhen 518083, China
2 Department of Otolaryngology, Head and Neck Surgery, Second Hospital, Shanxi Medical University, Taiyuan 030001, China
3 Shanxi Medical University-BGI Collaborative Center for Future Medicine, Shanxi Medical University, Taiyuan 030001, China
∗ Corresponding author yinjianhua@genomics.cn
∗∗ Corresponding author xjment@126.com
∗∗∗ Corresponding author liuchuanyu@genomics.cn
4 These authors contributed equally

5 Technical contact

6 Lead contact

07 9 2024
20 9 2024
07 9 2024
5 3 103298© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Examining nasal mucosa samples is crucial for nasal cavity disease research and diagnosis. Simultaneously obtaining high-quality data for single-cell transcriptomics (single-cell RNA sequencing [scRNA-seq]) and epigenomics (single-cell assay for transposase-accessible chromatin using sequencing [scATAC-seq]) of nasal mucosa tissues is challenging. Here, we present a protocol for processing human nasal mucosa samples to obtain data for both scRNA-seq and scATAC-seq. We describe steps for extracting human nasal mucosa tissue, mechanical and enzymatic dissociation, lysis of red blood cells, and a viability assay. We then detail procedures for library preparation and quality control.

Graphical abstract

Highlights

• Guidance on single-cell dissociation and nuclei extraction from human nasal mucosa samples

• Details for obtaining high-viability cell suspension and high-quality nuclei suspension

• High-throughput library preparation for both scRNA-seq and scATAC-seq

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Examining nasal mucosa samples is crucial for nasal cavity disease research and diagnosis. Simultaneously obtaining high-quality data for single-cell transcriptomics (single-cell RNA sequencing [scRNA-seq]) and epigenomics (single-cell assay for transposase-accessible chromatin using sequencing [scATAC-seq]) of nasal mucosa tissues is challenging. Here, we present a protocol for processing human nasal mucosa samples to obtain data for both scRNA-seq and scATAC-seq. We describe steps for extracting human nasal mucosa tissue, mechanical and enzymatic dissociation, lysis of red blood cells, and a viability assay. We then detail procedures for library preparation and quality control.

Subject areas

Cell isolation
Single Cell
Cell separation/fractionation
Genomics
Sequencing
==== Body
pmcBefore you begin

Institutional permissions

Human tissue samples used for scientific study should be obtained from volunteers or known patients, and official ethical approval from relevant authorities. The human nasal mucosa tissue samples used in this experiment were obtained from the Department of Otolaryngology, Second Hospital of Shanxi Medical University, with informed consent and approval from the BGI Ethics Committee (license number BGI-IRB 24040).

Preparation before the experiment

Timing: Days to hours before the experiment

1. Prepare all enzymes, buffers, culture media, solutions, and consumables that can be prepared in advance. Please refer to the key resources table and the materials and equipment section for detailed information.

2. Surgically collected nasal mucosa tissue can be stored in tissue preservation solution at 4°C for up to 72 h. Collection and transportation should be done before the start of the experiment.

Preparation on the day of the experiment

Timing: 30 min

3. Disinfect 2 sets of tweezers and ophthalmic scissors with 75% alcohol, soak for 10 min, and then rinse twice with distilled water.

4. Place cleaned tweezers, Petri dishes, scissors, pipette tips, pipettes, cell strainers with pore sizes of 40 μm, 50 mL tubes, 15 mL tubes, 1.5 mL tubes, ice bucket, and trash bin on a clean bench. Switch on the UV lamp to disinfect the work bench for 30 min prior to use.

5. Prepare the centrifuge and water bath. Set the centrifuge at 4°C and Add distilled water to the marked line in the water bath shaker and set the temperature to 37°C.

6. Thaw enzyme stock solutions (refer to materials and equipment), fetal bovine serum (FBS) (refer to materials and equipment) on ice. All other reagents stored at −20°C should be thawed and placed on ice for use.

7. Prepare reagents that need to be freshly prepared on the day of the experiment, and place them on ice after preparation for immediate use.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Biological samples	
	
Nasal mucosa of human	The Second Hospital of Shanxi Medical University	-	
	
Chemicals, peptides, and recombinant proteins	
	
Deoxyribonuclease I, 100 mg	Worthington	LS002139	
Liberase TM Research Grade	Roche	5401127001	
RPMI medium 1640	Gibco	11875093	
Fetal bovine serum (FBS)	EBT	XQ-1010	
Phosphate-buffered saline (PBS)	Gibco	14190144	
Acridine orange/propidium iodide (AO/PI) solution	Countstar	RE010212	
DAPI	Beyotime	C1006	
Bovine serum albumin (BSA)	BBI	A600332-0005	
Red blood cell lysis buffer	Solarbio	R1010	
DMSO	Sigma	D5879	
Digitonin	Sigma	D141	
37% Formaldehyde solution	Aladdin	F111936-500mL	
Glycine	BBI	A100167-0100	
1 M Tris pH 8.0	Thermo Fisher Scientific	AM9855G	
HEPES	BBI	A600264-0050	
Sucrose	Sigma	S7903	
1 M KCl	Thermo Fisher Scientific	AM9640G	
1 M MgCl2	Thermo Fisher Scientific	AM9530G	
100 mM DTT	Thermo Fisher Scientific	18064014	
Protease inhibitor cocktail	Roche	04693116001	
10% NP 40	Roche	11332473001	
10% Tween 20	Thermo Fisher Scientific	28320	
RNase inhibitor (40 U/μL)	BGI	01E0109MM	
Ambion nuclease-free water (NF-H2O)	Invitrogen	AM9932	
MGIEasy tissue storage reagent	MGI	940-001755-00	
	
Critical commercial assays	
	
DNBelab C series high-throughput single-cell RNA library preparation set V2.0	MGI	940-000519-00	
DNBelab C series high-throughput single-cell ATAC library preparation set	MGI	940-000793-00	
	
Other	
	
Petri dishes	Falcon	351006	
Pipette tips, 10 μL	Axygen	T-300-L-R-S	
Pipette tips, 200 μL	Axygen	T-200-C-L-R-S	
Pipette tips, 1000 μL	Axygen	T-1000-C-L-R-S	
Pasteur pipette	BBI	F621007-0001	
1.5 mL microcentrifuge tube	VIOX	V1501-C	
C-Chip hemocytometer	INCYTO	DHC-N01	
15 mL centrifuge tube	Corning	430052	
50 mL centrifuge tube	CORNING	430828	
0.22 μm syringe filter	Millipore	SLGP033R	
Cell strainer, 40 μm	Falcon	352340	
Countstar chamber slide	Countstar	CO010101	
Sealing film	BIOFOUNT	WF-666M	
Centrifugal machine	Eppendorf	5910Ri	
Water bath shaker	MIULAB	SWT-100	
Countess II FL automatic cell counter	Countstar	RY082K2001	
Bio-fragment analyzers	Agilent 2100 Bioanalyzer	G2939BA	
Fluorescence microscope	Olympus	IX71	
Clean bench	AIRTECH	SW-CJ-2FD	
Note: All reagents and instruments are interchangeable. They must be of the same quality and functionality.

Materials and equipment

Liberase TM stock solution

Reagent	Final concentration	Amount	
Liberase TM	5 mg/mL	50 mg	
PBS	-	10 mL	
Total	10 mL	
Note: Take 500 μL of the equal solution and store in 1.5 mL microcentrifuge tube. It can be stored at −20°C for 6 months. Avoid repeated freezing and thawing of enzyme solutions to ensure enzyme activity.

DNase I stock solution

Reagent	Final concentration	Amount	
DNase I	4 mg/mL	100 mg	
0.15 M NaCl Solution	-	25 mL	
Total	25 mL	
Note: Take 500 μL of the equal solution and store in 1.5 mL microcentrifuge tube. It can be stored at −20°C for 6 months. Avoid repeated freezing and thawing of enzyme solutions to ensure enzyme activity.

1% Digitonin stock solution

Reagent	Final concentration	Amount	
Digitonin	1%	100 mg	
DMSO	-	5 mL	
NF-H2O	-	5 mL	
Total	10 mL	
Note: Dissolve Digitonin in DMSO first, then dilute it with NF-H2O to make a 1% Digitonin solution. Aliquot into 15 μL per tube, and store at −20°C for up to 1 year.

Digestive solution

Reagent	Final concentration	Amount	
DNase I stock solution	0.05 mg/mL	62.5 μL	
Liberase TM stock solution	125 μg/mL	125 μL	
RPMI MEDIUM 1640	-	4812.5 μL	
Total	5000 μL	
Note: Please prepare Digestive solution immediately before use and place it on ice before use.

Stop solution

Reagent	Final concentration	Amount	
FBS	2%	0.4 mL	
RPMI MEDIUM 1640	-	19.6 mL	
Total	20 mL	
Note: It can be stored at 4°C for up to 7 days.

10% BSA in PBS

Reagent	Final concentration	Amount	
BSA	10%	1 g	
1x PBS	-	10 mL	
Total	10 mL	
Note: It should be filtered by 0.22 μm syringe filter before storage, and stored at −20°C for up to 6 months.

0.04% BSA in PBS

Reagent	Final concentration	Amount	
10% BSA	0.04%	40 μL	
1x PBS	-	9960 μL	
Total	10 mL	
Note: It can be stored at 4°C for up to 5 days.

1% BSA in PBS

Reagent	Final concentration	Amount	
10% BSA	1%	500 μL	
1x PBS	-	4500 μL	
Total	5 mL	
Note: It can be stored at 4°C for up to 5 days.

0.1% formaldehyde solution

Reagent	Final concentration	Amount	
37% formaldehyde solution	0.1%	2.7 μL	
1x PBS		997.3 μL	
Total		1 mL	
Note: Please prepare 0.1% formaldehyde solution immediately before use.

0.25 M glycine solution

Reagent	Final concentration	Amount	
2.5 M glycine solution	0.25 M	100 μL	
1x PBS		900 μL	
Total		1 mL	
Note: It should be filtered by 0.22 μm syringe filter before storage.

NIM Buffer

Reagent	Final concentration	Amount	
1 M Tris pH 8.0	10 mM	500 μL	
Sucrose	250 mM	4.3 g	
1 M KCl	25 mM	1.24 mL	
1 M MgCl₂	5 mM	250 μL	
NF-H₂O	-	Up to 50 mL	
Total		50 mL	
Note: It should be filtered by 0.22 μm syringe filter before storage. It can be stored at 4°C for up to 15 days.

Homogenization Buffer

Reagent	Final concentration	Amount	
NIM pH 8.0	-	427 μL	
100 mM DTT	0.1 mM	0.5 μL	
100x Protease inhibitor cocktail	1x	5 μL	
RNase inhibitor (40 U/μL)	0.2 U/μL	2.5 μL	
10% NP 40	0.1%	5 μL	
1% Digitonin	0.01%	5 μL	
10% BSA in PBS	1%	50 μL	
10% Tween 20	0.1%	5 μL	
Total		500 μL	
Note: Please prepare Homogenization Buffer immediately before use and place it on ice before use.

Homogenization Buffer-Washing

Reagent	Final concentration	Amount	
NIM pH 8.0	-	874 μL	
100 mM DTT	0.1 mM	1 μL	
100x Protease inhibitor cocktail	1x	10 μL	
RNase inhibitor (40 U/μL)	0.2 U/μL	5 μL	
10% Tween 20	0.10%	10 μL	
10% BSA in PBS	1%	100 μL	
Total		1 mL	
Note: Please prepare Homogenization Buffer-Washing immediately before use and place it on ice before use.

Step-by-step method details

Tissue dissociation

Timing: 60 min

1. Sample preparation.a. Move the tissue from the MGIEasy Tissue Storage Reagent to a 5 cm petri dish containing 2 mL of PBS.

b. Rinse twice, using 1 mL of PBS for each rinse.Note: If the tissue is flocculent, centrifuge the tissue in the tissue preservation solution, 400 g for 5 min at 4°C.

c. Measure the volume and mass of the tissue (Figure 1).Note: MACS Tissue Storage Solution (130-100-008) from Miltenyi Biotec is also recommended here to preserve isolated fresh tissue samples in 48 h to maintain the cell activity and stabilize the gene expression levels.

Figure 1 Measurement of nasal mucosa tissue

(A) The length of sample.

(B) The width of sample.

(C) The weight of sample.

2. Place the tissue in a sterile 5 cm petri dish and keep it moist with PBS. Carefully remove the nasal cartilage using tweezers and scissors (Figure 2A). Use forceps to gently transfer the tissue to a 1.5 mL microcentrifuge tube, add 200 μL of digestive solution.Figure 2 Mechanical dissociation of nasal mucosa sample

(A) Removal of cartilage from nasal mucosa tissue.

(B) Shearing of nasal mucosa tissue using scissors.

3. Cut the tissue with scissors into small pieces, approximately 2–4 mm3 in size (Figure 2B).

CRITICAL: Keep the tissue moist at any time during mechanical separation. Ensure that the tissue is immersed in the buffer to prevent dehydration from affecting the viability of cells after digestion.

4. Transfer chopped tissue.a. Transfer the chopped tissue from 1.5 mL tube to the 15 mL centrifuge tube using a Pasteur pipette.

b. Rinse the tissue with 1 mL digestive solution in 1.5 mL centrifuge tube, and transfer to the 15 mL centrifuge tube (collect as many cells as possible).

c. Add the rest of the digestive solution to 15 mL centrifuge tube for a total digestible volume of 5 mL.

Note: If the measured original mass of tissue after removal of cartilage is greater than 0.2 g, divide it into two tubes for digestion, using 5 mL digestive solution per tube.

CRITICAL: Retain the Pasteur pipette used for the first transfer and reuse it in subsequent rinses. Transfer any small tissue fragments that remain adhered inside the pipette from the first transfer into a 15 mL tube. Make sure all the tissue pieces have been collected and transferred.

5. Wrap the tube cap with 2 sheets of sealing film to prevent leakage during shaking. The sealing film is used in addition to the cap to provide additional protection against leaks.

6. Place the tubes in a water bath shaker, secure horizontally with a stationary band, and digest for 40 min at 37°C, shaking speed 60 rpm. During the shaking, make sure that the tissue fragments are moving horizontally (Figure 3).Figure 3 Enzymatic dissociation

(A) Nasal mucosa tissue was put into a water bath shaker for digestion.

(B) Nasal mucosa tissue before digestion.

(C) Nasal mucosa tissue after digestion.

7. Terminate digestion and filter cell suspension (Figures 4A and 4B) (troubleshooting 1).a. Remove the tube from the water bath shaker and place it on bench.

b. Add 5 mL stop solution to the tube.

c. Place a 40 μm cell filter on a 50 mL centrifuge tube.

d. Carefully pour the digested cell suspension into the cell filter.

e. The tube containing digested cell suspension was rinsed with 2 mL stop solution, and then the rinsed buffer was added to the same cell filter to continue collecting filtrate.

f. Finally, use the pipette to add a further 2 mL of the stop solution to the cell filter and rinse the filter.

g. Continue collecting filtrate to the same 50 mL centrifuge tube until no more liquid passes through.

Figure 4 Filtration of the cell suspension after the digestion reaction

(A and B) Filtration of cell suspension.

(C) Cell suspension after filtration.

(D) Cell suspension after centrifugation, where a red precipitate is clearly visible.

CRITICAL: The digested cell suspension must be poured slowly into the cell strainer, ensuring that the liquid level does not rise above the surface of the strainer. Allow the filtrate to slowly pass through before continuing to pour more suspension.

8. Centrifuge cell suspension filtered at 4°C with 400 g for 5 min (Figures 4C and 4D).

9. Gently resuspend the cell pellet using the 1 mL of 0.04% BSA PBS.

10. Measure cell viability and concentration.a. Take 10 μL of cell suspension at this point, dilute it to the appropriate concentration, and count it after 1:1 dilution with AO/PI Stain: A volume of 10 μL of AO/PI Stain add to the PCR tube, followed by the introduction of 10 μL of cell suspension.

b. The solution should then be mixed for a total of 15 cycles.

c. A volume of 20 μL of the resultant mixture should be transferred to a new chamber of the Countstar chamber slide in order to determine the viability of the cells.

d. Record the viability and concentration of cells suspension (Figure 5) (troubleshooting 2).Figure 5 Picture of cells stained by AO/PI solution

CRITICAL: If the total number of cells is less than 100,000, removal of red blood cells is not recommended, because the removal of red blood cells may also result in the loss of some nucleated cells.

Red blood cell lysis

Timing: 35 min

11. Perform the following red blood cell lysis if a distinct red coloration of the cell pellet is observed (Figure 6);a. Perform red blood cell lysis on the cell suspension from step 9.

Add 3 mL of Red Blood Cell Lysis Buffer (the volume of red blood cell lysis buffer added is 3 times the volume of the cell suspension) and gently pipette up and down to mix the cells.Note: In addition to Red Blood Cell Lysis Buffer from Solarbio, red blood cell lysis buffers from other vendors with the same function are also workable. Red blood cell lysis solution (10x) from Miltenyi Biotec (130-094-183) is also recommended here; find the protocol via IM0001772.PDF (miltenyibiotec.com).

b. Place on ice for 15 min;

c. Add 3 mL of 0.04% BSA PBS, invert and mix.

d. Centrifuge the cells at 400 g for 8 min at 4°C, remove the supernatant and keep the cell pellet.

Figure 6 The process of red blood cell lysis

(A) Addition of Red Blood Cell Lysis Buffer to the cell suspension.

(B) Incubation on ice.

(C) After centrifugation, a significant decrease in red blood cell pellet was observed.

(D) Resuspended cell suspension after red blood cell lysis.

12. Add appropriate amount of 0.04% BSA PBS according to the amount of precipitate in the tube. Resuspend and pipette up and down 15 times.

13. Live cell microscopy. Take 10 μL of AO/PI staining solution into the PCR tube, and add 10 μL of cell suspension and mix 15 times. Load 20 μL of mixture to a new chamber of Countstar chamber slide for cell viability determination. Record the viability and concentration of cells suspension after red blood cell lysis (Figure 7).Figure 7 Picture of cells suspension after red blood cell lysis stained by AO/PI solution

14. If cell viability is greater than 80%, subsequent experiments can be performed. If the cell viability is less than 80%, the dead cell removal experiment needs to be performed (troubleshooting 3) (troubleshooting 4).

Note: Cell suspension perform follow-up procedures immediately. If conditions do not allow, follow-up can be performed if cell suspension viability remains above 80% within 4 h at 4°C.

Nuclear preparation for single-cell ATAC-seq

Cell fixation

Timing: 20 min

15. Take 500,000 cells in 0.04% BSA PBS resuspended cells from step 9 in a 1.5 mL tube. (Record the actual quantity if less than 500,000 cells.).

16. Centrifuge at 4°C, 1000 g for 5 min, and slowly remove the supernatant.

17. Resuspend the cell nuclei pellet in 150 μL 0.1% formaldehyde in the same 1.5 mL tube. Incubate on ice for 5 min.

CRITICAL: Due to the toxicity of formaldehyde, it should be used under a chemical mask.

18. Terminate fixation by adding 150 μL of 0.25 M glycine solution, incubate on ice for 5 min, centrifuge at 1000 g for 3 min at 4°C, and discard supernatant.

19. Add 500 μL of 1% BSA in PBS, centrifuge at 1000 g for 3 min at 4°C, discard the supernatant.

20. Repeat step 19 once. Proceed to the lysis step.

Nuclei extracted from cell suspension

Timing: 20 min

21. Take 100 μL of pre-cooled Homogenization Buffer to resuspend the cells. Gently mix 30 times, incubate on ice for 3 min.

22. Add 480 μL of pre-cooled Homogenization Buffer-washing to the lysed cell suspension, slowly mix 5 times

CRITICAL: If the number of cells before cell fixation is less than 200,000 cells, the volume of Homogenization Buffer-washing should be reduced to 200 μL).

23. Centrifuge the cells at 500 g for 5 min at 4°C. Discard the supernatant and wash the nuclei again using 480 μL of pre-cooled Homogenization Buffer-washing.

CRITICAL: If the number of cells before cell fixation is less than 200,000 cells, the volume of Homogenization Buffer-washing should be reduced to 200 μL).

24. Centrifuge the cells at 500 g for 5 min at 4°C.

25. Resuspend the nuclei with 50 μL 1% BSA PBS, gently mix (use 20 μL 1% BSA PBS directly to resuspend the nuclei when the number of cells is less than 500,000).

26. Count the concentration of nuclei by DAPI staining. Take 18 μL of 1% BSA PBS into a new PCR tube, add 2 μL of well-mixed nuclei, and add 20 μL of DAPI to gently blow and mix.

27. Take 10 μL of DAPI-stained nuclei to a new chamber of C-Chip hemocytometer, taking care not to produce air bubbles.

28. Observe the counting area of the cell counter chip under a fluorescence microscope. Count the nuclei of cells in the field of view that are stained with blue fluorescence (Figure 8).Figure 8 Microscopic examination of nuclei extracted from cell suspension

(A) Bright field.

(B) After DAPI staining.

Density(cellnumber/μL)=totalnumberofnucleiinthefourlargesquareareasN/4×10×dilutionn.

29. Nuclei QC standard: the proportion of adherent nuclei is less than 10%; the edges of the fluorescent spots are clear and rounded under the microscope; the nuclei have a regular shape and do not exhibit irregular morphology; the fluorescence concentration is not diffuse.

Note: Nuclei suspension perform follow-up procedures immediately. If conditions do not allow, follow-up can be performed if nuclei suspension stored within 4 h at 4°C.

Single-cell RNA-seq and ATAC-seq library construction

Timing: 12 h

30. Immediately follow the instructions of DNBelab C Series High-throughput Single-cell RNA Library Preparation Set V2.0 for library construction after obtaining the single-cell suspension in step 12 (troubleshooting 5). Find the protocol via 63774a7a00800.pdf (mgi-tech.com).

31. Immediately follow the instructions of DNBelab C Series High-throughput Single-cell ATAC Library Preparation Set V1.0 for the construction of the library, after acquisition of the single-cell nuclei suspension detailed in step 25. The instructions can be found in the document 661c959c98114.pdf, accessible via the website mgi-tech.com.Figure 9 Typical product and library fragment size distribution

(A) The fragment size distribution of the cDNA product.

(B) The fragment size distribution of cDNA library.

(C) The fragment size distribution of the ATAC product.

Note: Other platforms can be chosen for scRNA-seq and scATAC-seq library construction.

CRITICAL: Two quality control (QC) checks are required at this step to assess the fragment size distributions of the cDNA product and library. The peak should be between 500 bp and 2000 bp in the fragment size distribution of the cDNA products. The cDNA library should be between 350 bp and 550 bp. Figures 9A and 9B show typical fragment size distributions of cDNA products and libraries. ATAC library product fragment size distribution generally shows an enriched peak at around 200 bp, with reference distribution shown in Figure 9C (troubleshooting 6).1

Note: Users can choose other platforms for fragment size distribution determination.

Expected outcomes

Using this protocol, a single-cell suspension with a total cell counts exceeding 500,000 and a viability rate higher than 85% can be obtained from tissues weighing more than 0.05 g. This digestion method is also applicable to the digestion of other mucosal tissues. The method can achieve high-quality single-cell RNA-seq and single-cell ATAC-seq data. Using this method for single-cell digestion, we obtained a variety of cell subpopulations with diverse gene expression profiles, including epithelial cells, fibroblasts, glandular cells, endothelial cells, and immune cells (such as T cells, B cells, myeloid cells, and mast cells), demonstrating the genetic heterogeneity within the population.2 Typically, samples rich in immune cells pose challenges for single-cell ATAC-seq library construction, but this method optimizes the nuclei extraction process and significantly improves the data quality of single-cell ATAC-seq upon testing.

Limitations

When following standard operating procedures, ensuring consistency in instrument and reagent batches is a priority. However, batch effects can still arise due to the pathological complexity of clinical samples and variations in the duration, force, and environmental factors influencing different personnel conducting the same procedure.3 Given the inherent heterogeneity of pathological tissues, drawing conclusions about changes in specific cell proportions between different samples requires appropriate control sample data and a statistically significant sample size. Different single-cell platforms may also exhibit biases in capturing different types of cells. Therefore, comparing results from different platforms necessitates the use of data generated from the same single-cell platform, supplemented with additional support from spatial transcriptomics data or flow cytometry analysis.4

Troubleshooting

Problem 1

Large tissue chunks remain after digestion, blocking the cell strainer during filtration.

Potential solution

• The tissue chunks were not adequately minced. If the tissue pieces are too large, place the tissue in a 10 cm culture dish, add an appropriate amount of digestion buffer to immerse the tissue, and then use a scalpel to mince the tissue repeatedly in a crisscross pattern. Aim to mince the tissue into pieces around 2–4 mm³ in size.

• The digestive enzymes may have been inactivated. Enzymes should be dissolved and stored according to the conditions specified in the instructions, and prepared to the target concentration. When using the enzyme stock solution, thaw and handle it on ice, then return it to −20°C for storage. Avoid more than three freeze-thaw cycles for each aliquot.

• If the cell strainer becomes clogged and the digestion buffer cannot filter through, stop pouring the cell suspension, rinse the strainer with stop solution, and replace it with a new strainer to continue filtering the remaining cell suspension.

Problem 2

Lower-than-expected total cell count after digestion.

Potential solution

Potential reasons include inadequate mincing of the tissue or insufficient enzyme activity, as previously discussed. Another reason could be the loss of tissue or cells during the procedure. Ensure that every transfer step is performed carefully to maximize the transfer of all tissues and cells. If tissue or cells are not fully recovered due to adherence, consider using low-adhesion pipette tips and centrifuge tubes.

During cell counting, make sure to thoroughly mix the cell suspension. After adding AO/PI staining solution, mix thoroughly again before loading onto the counting chamber.

Problem 3

Low cell viability after digestion.

Potential solution

• Store the tissue in MGIEasy Tissue Storage Reagent at 4°C for transportation and storage, and ideally, keep the storage time under 48 h before starting the experiment. Ensure that the tissue is not allowed to dry out during measurement and other handling processes.

• During cell handling, such as pipetting and transferring tissue and cell suspensions, use a Pasteur pipette or wide-bore pipette tips to minimize mechanical damage. For counting, use regular pipette tips and handle the suspension gently and slowly.

• If low cell viability (below 80%) is intrinsic to the sample, remove dead cells with a Dead Cell Removal Kit from Miltenyi Biotec (130-094-183). Find the protocol via IM0001772.PDF (miltenyibiotec.com).

Note: MojoSort Human Dead Cell Removal Kit from BioLegend (480159) is also recommended here for dead cell removal. Find the protocol in the website (https://www.biolegend.com/en-gb/protocols/mojosort-dead-cell-removal-protocol).

Problem 4

High cell clumping.

Potential solution

For cell pellet resuspension before counting: use regular pipette tips, ensuring that the resuspension volume is less than the pipette’s capacity and pipette up and down 15 times. Then, add more resuspension buffer and mix gently by inversion. And, excessive free DNA causes clumping, increase the concentration of DNase I to address this issue.5

Problem 5

Low number of cells obtained for scRNA-seq.

Potential solution

• Possible reason 1: Inaccurate cell concentration during counting. If cell concentration exceeds 3000 cells/μL, sampling accuracy may be compromised. Dilute the cell concentration to between 700–2000 cells/μL, then recount. The best practice is to count three times and take the average. If the cell suspension concentration is below 500 cells/μL, reduce resuspension volume after centrifugation and recount.

• Possible reason 2: Single-cell microfluidic chip clogging, which could be due to impurities introduced during handling or large cell clumps.

Problem 6

Unexpected fragment size distribution of cDNA product or library.

Potential solution

• If the cDNA fragment size distribution peak is below 700 bp, it may indicate mRNA degradation. Store surgical samples in a preservation solution at 4°C as quickly as possible, ensuring full immersion during transport, and preventing tissue exposure to air. Keep samples moist during weighing and cutting, using PBS if necessary. Proceed with single-cell experiments as soon as the cell suspension is prepared, ideally within 2 h, and keep the suspension on ice during this period.

• If the cDNA fragment size shows many peaks above 2000 bp or lacks a clear peak, genomic contamination may be the cause. This rare issue can result from unusually low mRNA abundance or poor cell viability. Samples with cell viability below 80% should undergo dead cell removal before use.

• For small library fragments or low library concentration, the issue could be excessive enzyme use during fragmentation. When handling the fragmentation enzyme, ensure the pipette tip remains at the surface of the enzyme solution to prevent excess enzyme from adhering to the outer surface of the tip. Perform fragmentation on ice to prevent premature enzyme activity.

Resource availability

Lead contact

Please contact Dr. Chuanyu Liu (liuchuanyu@genomics.cn) for further information and requests for resources and reagents.

Technical contact

Please contact the technical contact, Yaling Huang (huangyaling@genomics.cn), with any technical questions regarding this protocol.

Materials availability

No new unique reagents or materials were generated in this study.

Data and code availability

The datasets supporting the current study have not been deposited in a public repository because the paper containing these datasets have not been published yet, but a sample raw dataset associated to this protocol are available from the corresponding author on request.

Acknowledgments

This research was supported by the Shenzhen Key Laboratory of Single-Cell Omics (no. ZDSYS20190902093613831 ).

Author contributions

Y.H., Y.W., and S.H. developed the protocol with advice from J.Y. Y.H. and Q.W. wrote the manuscript with help from G.C. Y.W., G.C., and Q.W. performed the experiment with assistance from Z.L., S.G., X.H., and Y.L. C.L., J.X., and J.Y. supervised the study and revised the manuscript. All the authors reviewed and approved the final manuscript.

Declaration of interests

The authors declare no competing interests.
==== Refs
References

1 Buenrostro J.D. Giresi P.G. Zaba L.C. Chang H.Y. Greenleaf W.J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position Nat. Methods 10 2013 1213 1218 24097267
2 Wang W. Xu Y. Wang L. Zhu Z. Aodeng S. Chen H. Cai M. Huang Z. Han J. Wang L. Single-cell profiling identifies mechanisms of inflammatory heterogeneity in chronic rhinosinusitis Nat. Immunol. 23 2022 1484 1494 36138182
3 Čuklina J. Pedrioli P.G.A. Aebersold R. Review of Batch Effects Prevention, Diagnostics, and Correction Approaches Methods Mol. Biol. 2051 2020 373 387 31552638
4 Ziegenhain C. Vieth B. Parekh S. Reinius B. Guillaumet-Adkins A. Smets M. Leonhardt H. Heyn H. Hellmann I. Enard W. Comparative Analysis of Single-Cell RNA Sequencing Methods Mol. Cell 65 2017 631 643.e4 28212749
5 Montanaro L. Poggi A. Visai L. Ravaioli S. Campoccia D. Speziale P. Arciola C.R. Extracellular DNA in biofilms Int. J. Artif. Organs 34 2011 824 831 22094562
