基于自定义多重标记策略的高级单细胞 RNA 测序（scRNA-seq）方案

Feng Gao; Xujie Liu; Fan Sun; Yadong Xiao; Gutian Xiao; Zhaoxia Qu

doi:10.21769/BioProtoc.5678

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Peer-reviewed

An Advanced Single-Cell RNA Sequencing (scRNA-seq) Protocol Utilizing Custom-Designed Multiplexing

基于自定义多重标记策略的高级单细胞 RNA 测序（scRNA-seq）方案

ZQ Zhaoxia Qu email

发布: 2026年05月05日第16卷第9期 DOI: 10.21769/BioProtoc.5678 浏览次数: 885

评审: Sébastien GillotinEVANGELOS THEODOROUAnonymous reviewer(s)

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Nov 2025

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Abstract

While cell hashing enhances single-cell RNA sequencing (scRNA-seq) efficiency and minimizes batch effects, commercial mouse hashtags often fail in FVB/N and several other strains due to antibody-epitope incompatibility. We describe a robust alternative utilizing biotinylated antibody cocktails and streptavidin-conjugated oligos to enable reliable sample multiplexing. This approach was validated in FVB/N lung tissues, yielding high-quality single-cell libraries. Our protocol offers a practical solution for researchers requiring strain-specific or custom-designed multiplexing strategies for single-cell transcriptomics.

Key features

• Strain-specific compatibility: Resolves the known H-2^q haplotype mismatch in FVB/N mice that fails standard commercial MHC-I hashtag antibodies in cell hashing.

• Multi-omic 5′ workflow integration: Enables simultaneous sample multiplexing with 10× Genomics 5 chemistry, facilitating joint gene expression and V(D)J repertoire (TCR/BCR) profiling.

• Enhanced non-immune cell labeling: Incorporates anti-CD326 (Ep-CAM) to ensure robust hashing of epithelial and tumor cells that may exhibit MHC-I downregulation or lack CD45.

• Customizable biotin-streptavidin framework: Provides a modular system using biotinylated antibody cocktails and streptavidin-barcodes, adaptable for any mouse strain or tissue-specific cell markers.

Keywords: scRNA-seq (单细胞 RNA 测序)

Cell hashing (细胞哈希标记)

Sample multiplexing (样本多重标记)

FVB/N mice (FVB/N 小鼠)

MHC-I haplotype mismatch (MHC-I 单倍型不匹配)

5′ gene expression (5′ 基因表达)

Biotin-streptavidin conjugation (亲和素-生物素偶联)

Graphical overview

Single-cell RNA sequencing (scRNA-seq) protocol utilizing custom-designed multiplexing

Background

Since its inception in 2009, single-cell RNA sequencing (scRNA-seq) has become an essential technology for profiling the transcriptomic heterogeneity of complex multicellular systems [1]. However, when multiple biological samples are processed in parallel, technical challenges arise, including limited throughput, prohibitive reagent costs, and potential batch effects. Sample multiplexing via cell hashing has emerged as a robust solution to these challenges [2–6]. By labeling individual samples with unique molecular barcodes before pooling and compartmentalization, researchers can integrate multiple samples into a single run, significantly reducing costs while enabling the computational identification and removal of cell doublets or multiplets.

Cell hashing is typically achieved by labeling cell surfaces with oligonucleotide-barcoded reagents, such as oligo-conjugated antibodies targeting ubiquitous surface proteins or lipid/cholesterol-modified oligonucleotides (LMO/CMO) that anchor into the plasma membrane [2–6]. For mouse studies, antibody-based hashing often relies on commercial TotalSeq reagents from BioLegend, which utilize a cocktail of anti-CD45 and anti-MHC Class I (M1/42) antibodies. However, these “universal” reagents are incompatible with the FVB/N and several other mouse strains. Specifically, the M1/42 clone in the commercial mix lacks reactivity with the H-2^q haplotype found in FVB/N mice, leading to labeling failure in non-immune (CD45-) populations. Furthermore, while alternative lipid-based multiplexing (e.g., 10× Genomics 3′ CellPlex) approaches are available, they are currently incompatible with single-cell 5′ chemistries, thereby precluding simultaneous profiling of T-cell and B-cell receptor (TCR/BCR) repertoires. The incompatibility of the M1/42 clone is not limited to the FVB/N strain; it also extends to other widely used models carrying the H-2^q, H-2^p, or H-2^r haplotypes, such as the NOD, BUB/BnJ, and RIIIS/J strains. In these genetic backgrounds, standard antibody-based multiplexing often results in the loss of signal in non-immune cell populations, a critical hurdle for lung research where epithelial-immune crosstalk is paramount.

To address these limitations, we developed a versatile cell hashing protocol optimized for the FVB/N strain and compatible with 10× Genomics 5′ workflows [7]. Our method utilizes a customized cocktail of biotin-conjugated antibodies, including anti-CD45 for immune cells, anti-MHC Class I (H-2^q) for broad nucleated cell coverage, and anti-CD326 (Ep-CAM) for epithelial and tumor cell enrichment, paired with sample-specific streptavidin-conjugated barcodes. This tripartite antibody approach ensures robust capture of both CD45⁺ immune infiltrates and CD45- parenchymal or malignant cells, the latter of which may exhibit MHC-I downregulation. Here, we provide a detailed, experimentally validated protocol for custom-designed multiplexing and cell hashing to enable high-resolution scRNA-seq on the lung tissues of wild-type (WT) or myeloid PDLIM2 deletion (mKO) FVB/N mice intratracheally instilled with lipopolysaccharide (LPS) or phosphate-buffered saline (PBS).

The choice of the PDLIM2-deficient model for this protocol validation is based on the gene’s critical role in lung homeostasis. PDLIM2 (PDZ-LIM domain-containing protein 2, also known as SLIM or mystique) is a ubiquitously expressed gene with the highest level in the lung [8–10]. At the cellular level, PDLIM2 is expressed abundantly in various epithelial and immune cells, particularly lung epithelial cells and myeloid cells, two functionally critical cell types in the lung [7,10–18]. Not surprisingly, PDLIM2 serves as a vital molecular checkpoint whose repression has been linked to various pathogenic conditions, especially in the lung, such as lung cancer, chronic obstructive pulmonary disease (COPD), interstitial lung disease (ILD)/pulmonary fibrosis (PF), lung infection, and infectious diseases [7,11–16,19–23]. Mechanistically, PDLIM2 acts as a novel ubiquitin ligase enhancer (E5) to stabilize and chaperone the E3 SCF^β-TrCP to promote the ubiquitination of nuclear RelA (the prototypical NF-κB member that is also known as p65) and possibly STAT3 and other proteins for proteasomal degradation [24–32]. NF-κB and STAT3 are physiologically vital transcription factors that are tightly regulated, and their persistent activation plays causative roles in various diseases, including lung cancer, COPD, ILD/PF, and infections [33–60]. While it complements existing approaches, the novel scRNA-seq using custom-designed multiplexing is far more advanced in studying NF-κB, STAT3, or any other genes and pathways [61–63].

Materials and reagents

Biological materials

1. Mice: PDLIM2^flox/flox/lysozyme M-Cre^+/- mice (mKO) mice were generated as previously described [11,14,16]. Wild-type (WT) control mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were maintained on a pure FVB/N genetic background. For the scRNA-seq experiment, one biological replicate was used per experimental condition (WT-Ctrl, WT-LPS, mKO-Ctrl, and mKO-LPS) and multiplexed into a single library using the described cell hashing protocol.

Reagents

1. Lung Dissociation kit, mouse (Miltenyi Biotec, catalog number: 130-095-927); contains buffer S (20× stock solution), enzyme D (lyophilized powder), and enzyme A (lyophilized powder)

Note: See Recipes for enzyme D and enzyme A preparation before use.

2. Dead Cell Removal kit (Miltenyi Biotec, catalog number: 130-090-101); contains dead cell removal microbeads and 20× binding buffer stock solution

3. Red blood cell lysis solution (10×) (Miltenyi Biotec, catalog number: 130-094-183)

4. Biotin anti-mouse CD45 (BioLegend, catalog number: 103103)

5. Biotin anti-mouse MHC Class I (eBioscience, catalog number: 13-5998-81; clone 34-1-2S), which specifically reacts with the H-2^q haplotype of FVB/N mice, as well as b, s, r, and p haplotypes

6. Biotin anti-mouse Ep-CAM (BioLegend, catalog number: 118203)

7. TotalSeq^TM-C0951 PE streptavidin (BioLegend, catalog number: 405261)

8. TotalSeq^TM-C0952 PE streptavidin (BioLegend, catalog number: 405263)

9. TotalSeq^TM-C0953 PE streptavidin (BioLegend, catalog number: 405265)

10. TotalSeq^TM-C0954 PE streptavidin (BioLegend, catalog number: 405267)

11. Ethanol (Decon Labs, catalog number: 2701)

12. RPMI-1640 medium (Lonza, catalog number: 12-702F)

13. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9625)

14. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541)

15. Disodium hydrogen phosphate heptahydrate (Na₂HPO₄·7H₂O) (Fisher Scientific, catalog number: BP331-500)

16. Potassium phosphate monobasic (KH₂PO₄) (Acros Organics, catalog number: 205925000)

17. Bovine serum albumin (BSA) (MP Biomedicals, catalog number: 199898)

18. Dual Index kit TT Set A 96 rxns (10× Genomics, Inc., catalog number: 1000215)

19. Dual Index kit TN Set A 96 rxns (10× Genomics, Inc., catalog number: 1000250)

20. Chromium Next GEM Single Cell 5’ kit v2, 4 rxns (10× Genomics, Inc., catalog number: 1000265)

21. Chromium Next GEM Chip K Single Cell kit, 16 rxns (10× Genomics, Inc., catalog number: 1000287)

22. 5’ Feature Barcode kit, 16 rxns (10× Genomics, Inc., catalog number: 1000256)

23. Trypan Blue (Sigma-Aldrich, catalog number: T8154)

24. Lipopolysaccharide (LPS) (Sigma-Aldrich, catalog number: L2880)

Solutions

1. Phosphate-buffered saline (PBS) (see Recipes)

2. Washing buffer (see Recipes)

3. Labeling buffer (see Recipes)

4. Buffer S (1× solution) (see Recipes)

5. Enzyme D (see Recipes)

6. Enzyme A (see Recipes)

7. Red blood cell lysis solution (1×) (see Recipes)

8. LPS solution (see Recipes)

Recipes

1. PBS

Reagent	Final concentration	Quantity or volume
NaCl	137 mM	8 g
KCl	2.7 mM	0.2 g
Na₂HPO₄·7H₂O	10 mM	2.16 g
KH₂PO₄	1.8 mM	0.24 g
H₂O		1,000 mL

To prepare 1 L of 1× PBS, begin by dissolving 8.0 g of NaCl, 0.2 g of KCl, 2.16 g of Na₂HPO₄·7H₂O, and 0.24 g of KH₂PO₄ in approximately 800 mL of distilled or Milli-Q water while stirring with a magnetic stir bar. Once the salts are completely dissolved, calibrate a pH meter and adjust the solution to pH 7.4 using a few drops of concentrated HCl (to lower pH) or NaOH (to raise pH), as needed. Finally, transfer the solution to a volumetric flask or graduated cylinder and add distilled water until the total volume reaches exactly 1 L. Then, filter-sterilize (0.22 μm) or autoclave the buffer to prevent microbial growth.

2. Washing buffer

Reagent	Final concentration	Quantity or volume
BSA	0.04%	0.2 g
PBS	1×	500 mL

3. Labeling buffer

Reagent	Final concentration	Quantity or volume
BSA	1%	2 g
PBS	1×	200 mL

4. Buffer S (1×)

Reagent	Final concentration	Quantity or volume
Buffer S (20×)	1x	2 mL
H₂O		38 mL

To prepare 40 mL of 1× buffer S, perform a 1:20 dilution by measuring 2 mL of 20× buffer S and transferring it into a clean container. Add 38 mL of sterile, distilled water to reach the final volume of 40 mL. Mix the solution thoroughly to ensure homogeneity and store the resulting 1× buffer at 2–8 °C until use.

5. Enzyme D

Reagent	Final concentration	Quantity or volume
Enzyme D	1×	1 vial
RPMI-1640		3 mL

Reconstitute the lyophilized enzyme D powder in each vial with 3 mL of RPMI-1640 medium. Close the vial and invert gently for 5 min. Prepare aliquots and store at -20 °C, avoiding freeze/thaw cycles. This solution is stable for 6 months after reconstitution.

6. Enzyme A

Reagent	Final concentration	Quantity or volume
Enzyme A	1×	1 vial
Buffer S (1×)		1 mL

Reconstitute the lyophilized enzyme A powder in the vial with 1 mL of 1× buffer S. Do not vortex. Prepare aliquots and store at -20 °C, avoiding freeze/thaw cycles. This solution is stable for 6 months after reconstitution.

7. Red blood cell lysis solution (1×)

Reagent	Final concentration	Quantity or volume
Red blood cell lysis solution (10×)	1×	5 mL
H₂O		45 mL

Dilute 5 mL of red blood cell lysis solution (10×) with 45 mL of sterile, distilled water.

8. LPS solution

Reagent	Final concentration	Quantity or volume
LPS	5 mg/mL	10 mg
PBS (1×)		2 mL

Stock solutions of LPS at 5 mg/mL are prepared, aliquoted, and frozen at -80 °C to help standardize instillations. Mice are intratracheally instilled with LPS at 4 mg/kg body weight.

Laboratory supplies

1. GentleMACS C tubes (Miltenyi Biotec, catalog number: 130-093-237)

2. Cell strainers, 40 μm (Fisher, catalog number: 22-363-547)

3. Cell strainers, 70 μm (Fisher, catalog number: 22-363-548)

4. Petri dish (Falcon, catalog number: 351029)

5. 35 mm cell culture dish (Greiner Bio-One, catalog number: 627160)

6. MACS MS column (Miltenyi Biotec, catalog number: 130-042-201)

7. 15 mL conical tubes (TH Geyer, catalog number: 7696714)

8. 50 mL conical tubes (Greiner Bio-One, catalog number: 227261)

9. 150 mL vacuum filtration devices, pore 0.22 μm (Jet Biofil, catalog number: FCF010004)

10. 500 mL vacuum filtration devices, pore 0.22 μm (Jet Biofil, catalog number: FPE204500)

11. 2 mL cryovials (Maxxline, catalog number: MLC2B)

12. 5 mL serological pipettes (Greiner Bio-One, catalog number: 606180)

13. 10 mL serological pipettes (Greiner Bio-One, catalog number: 607180)

14. 25 mL serological pipettes (Greiner Bio-One, catalog number: 760160-TRI)

15. Sterile PES syringe filters (Thermo Fisher Scientific, catalog number: 15206869)

16. 50 mL 3-part syringes (Chirana T. Injecta, catalog number: CH03050LL)

17. 10 μL pipette tips (TH Geyer, catalog number: 7695881)

18. 20 μL pipette tips (TH Geyer, catalog number: 7695882)

19. 200 μL pipette tips (TH Geyer, catalog number: 7695884)

20. 1,250 μL pipette tips (TH Geyer, catalog number: 7695887)

21. Countess cell counting chamber slides (Thermo Fisher Scientific, Invitrogen, catalog number: C10283)

Equipment

1. CO₂ chamber

2. Biosafety cabinet

3. Styrofoam board

4. Forceps (Roboz Surgical Instrument, catalog number: RS-5135)

5. Scissors (Roboz Surgical Instrument, catalog number: RS-6802)

6. GentleMACS^TM Octo Dissociator with Heaters (Miltenyi Biotec, catalog number: 130-096-427)

Note: Alternatively, a standard GentleMACS^TM Dissociator without heating units may be used; in this case, the programmed heating steps must be substituted with manual incubations in a 37 °C water bath for the durations specified in the lung dissociation protocol.

7. Centrifuge (Thermo Fisher Scientific, Thermo Scientific^TM, model: IEC CL40R, catalog number: 11210927)

8. MACS MultiStand (Miltenyi Biotec, catalog number: 130-042-303)

9. MiniMACS Separator (Miltenyi Biotec, catalog number: 130-042-102) or OctoMACS Separator (Miltenyi Biotec, catalog number: 130-042-109) for use with MACS MS columns

Note: For higher throughput processing, the QuadroMACS^TM Separator (supporting up to 4 columns) or the semi-automated MultiMACS^TM Cell 24 Separator Plus (supporting up to 24 columns) may be used as alternatives to accommodate larger sample batches.

10. Countess II FL automated cell counter (Invitrogen, catalog number: AMQAF1000)

11. Chromium Controller & Next GEM Accessory kit (10× Genomics, catalog number: 1000202 or 1000204)

12. Thermocycler (Thermo Fisher Scientific, catalog number: 4375786)

Software and datasets

1. Cell Ranger (v7.0): Used for primary data processing, including alignment, filtering, and UMI counting. Available from 10× Genomics (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)

2. Seurat (v5.0): An R package utilized for secondary analysis, including quality control, normalization, dimensional reduction, and clustering. Available via CRAN or GitHub (https://satijalab.org/seurat/)

3. Ingenuity Pathway Analysis (IPA, QIAGEN): Used for functional enrichment and metabolic pathway analysis of differentially expressed genes. Accessible via QIAGEN Digital Insights (https://digitalinsights.qiagen.com/products-overview/discovery-insights-portfolio/analysis-and-visualization/qiagen-ipa/)

4. Dataset availability: The raw and processed mouse scRNA-seq datasets generated in this study have been deposited in the NCBI Gene Expression Omnibus (GEO) and are publicly available under the accession number GSE249800

Procedure

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文章信息

稿件历史记录

提交日期: Feb 5, 2026

接收日期: Mar 23, 2026

在线发布日期: Apr 12, 2026

出版日期: May 5, 2026

版权信息

如何引用

Gao, F., Liu, X., Sun, F., Xiao, Y., Xiao, G. and Qu, Z. (2026). An Advanced Single-Cell RNA Sequencing (scRNA-seq) Protocol Utilizing Custom-Designed Multiplexing. Bio-protocol 16(9): e5678. DOI: 10.21769/BioProtoc.5678.