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

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

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

3. Labeling buffer

4. Buffer S (1×)

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

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

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×)

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

8. LPS solution

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

A. LPS intratracheal instillation in mice

1. Intratracheally instill 6–8-week-old mice with either LPS (4 mg/kg body weight) or an equivalent volume of sterile PBS (control) under light anesthesia. Monitor mice daily for changes in body weight and clinical signs of respiratory distress or systemic inflammation. Typically, symptoms are observed starting on day 2 post-instillation.

B. Preparation of single lung cell suspension

1. Euthanize the mouse by CO₂ inhalation in a CO₂ chamber.

Note: Researchers must strictly adhere to local institutional animal care and use committee (IACUC) guidelines and regional regulations, as CO₂ inhalation protocols may vary or be restricted in certain jurisdictions.

2. Bring the mouse to a biosafety cabinet and dampen it with 70% ethanol.

3. Place the mouse front side up on a dissecting Styrofoam board and fix the arms and legs with needles or tape [64].

4. Use scissors to make an incision in the skin from the abdomen to the neck and tear the skin with forceps to expose the thoracic cage and neck.

5. Carefully cut the ribs to expose the heart and lungs [65].

6. Dissect the lung and remove efferent and afferent blood vessels, trachea, and connective tissue from the lung.

7. Dissect the lung into individual lobes and thoroughly rinse them in a 60 mm × 15 mm Petri dish containing 8 mL of ice-cold PBS. Ensure each lobe is fully submerged during the wash to effectively remove residual blood and surface contaminants.

8. Prepare an enzyme mix by adding 2.4 mL of RPMI-1640 medium, 100 μL of enzyme D, and 15 μL of enzyme A into a 35 mm cell culture dish [66].

9. Transfer the lung lobes to the dish containing the enzyme mix and cut them into 1–2 mm pieces with scissors and forceps.

10. Transfer the tissue pieces and enzyme mix into the gentleMACS C tube (or a sterile 50 mL conical tube if using manual dissociation).

Notes:

1. If a gentleMACS Dissociator is unavailable, manual dissociation can be performed by mincing the tissue into finer pieces using sterile surgical blades, followed by incubation in a 37 °C water bath with constant agitation (e.g., using a magnetic stirrer or orbital shaker) to ensure efficient enzymatic digestion.

2. In case more than one sample from the same treatment group is used, samples from the same treatment group may be pooled before dissociation (see General note 1).

11. Tightly close the C tube and attach it upside down to the sleeve of the gentleMACS Octo Dissociator with Heaters.

Critical: The use of the heater attachment ensures optimal enzymatic activity and consistent tissue digestion; if a heater-equipped dissociator is unavailable, the tube may be incubated in a 37 °C water bath between dissociation programs (see General note 2).

12. Select the gentleMACS Program 37C_m_LDK_1. This automated program consists of a 34-min sequence involving standardized mechanical dissociation steps interspersed with 37 °C incubation periods to optimize enzymatic tissue digestion (see manufacturer’s specifications for the Lung Dissociation kit, Mouse).

13. After termination of the program, detach the C tube from the dissociator.

14. Centrifuge the C tube at 300× g for 30 s at room temperature and discard the supernatant.

15. Resuspend the sample in 10 mL of RPMI-1640 medium and filter the cell suspension through a 70 μm cell strainer placed over a 50 mL conical tube.

16. Wash the cell strainer with 10 mL of RPMI-1640 medium and combine the wash with the cell suspension from step B15.

17. Centrifuge the cell suspension at 300× g for 10 min at room temperature. Aspirate the supernatant completely.

18. Resuspend the cell pellet in 1 mL of 1× red blood cell lysis solution and incubate on ice for 10 min.

19. Add 10 mL of washing buffer to terminate lysis and centrifuge at 300× g for 10 min at 4 °C.

20. Resuspend the cell pellet in 10 mL of washing buffer and count cells using a Countess^TM II Cell Counter.

C. Cell hashing

1. Transfer the cell suspension containing 1 × 10⁷ cells to a 50 mL conical tube and centrifuge at 300× g for 10 min at 4 °C.

2. Prepare the antibody cocktail by adding 1 mL of labeling buffer and 2.5 μg of each biotinylated antibody per sample (anti-CD45, anti-EpCAM, and anti-MHC class I).

Critical: Ensure the anti-MHC class I antibody used (e.g., clone 34–1-2S) is specifically reactive with the H-2^q haplotype of the FVB/N mouse; standard commercial MHC-I clones (such as M1/42) lack this reactivity and will result in labeling failure of non-immune cells.

3. Resuspend the cell pellet in 1 mL of the antibody pool by gently pipetting up and down at least five times using a P1000 tip to ensure a homogeneous suspension. Incubate the mixture for 30 min at 4 °C with continuous gentle rotation (e.g., using a tube rotator at 10–20 rpm) to prevent cell settling and ensure uniform antibody binding.

4. Centrifuge samples at 400× g for 5 min at 4 °C. Carefully aspirate the supernatant using a P1000 pipette tip, holding the tube at an oblique angle to avoid disturbing the cell pellet. Resuspend the pellet in 1 mL of cold labeling buffer by gentle pipetting or flicking the tube. Repeat this wash process two additional times for a total of three washes.

5. Resuspend each cell pellet in 1 mL of labeling buffer containing 1.2 μg of the unique TotalSeq^TM-C oligo-barcoded streptavidin. Ensure a homogeneous suspension by gently pipetting with a P1000 tip at least five times. Incubate the suspension for 20 min at 4 °C with continuous gentle rotation to prevent cell settling and ensure uniform antibody binding.

Critical: Ensure each biological sample is labeled with a unique streptavidin-conjugated oligo barcode (see General note 3).

6. Centrifuge samples at 400× g for 5 min at 4 °C. Then, wash three times with 1 mL of labeling buffer as in step C4.

D. Dead cell removal

1. Prepare 1× binding buffer by diluting the 20× binding buffer stock solution (supplied with the Dead Cell Removal kit) with sterile, double-distilled water (ddH2O) at a 1:20 ratio.

2. Resuspend the cell pellet from step C6 in 100 μL of dead cell removal microbeads.

Critical: The volume of dead cell removal microbeads should be maintained as specified for any cell load up to 10⁷ cells (see General note 4).

3. Mix well by gentle pipetting and incubate for 15 min at room temperature (20–25 °C) under static conditions.

Critical: Do not use a rotator or vortex during this incubation. The microbeads are in a colloidal suspension and do not settle; therefore, active agitation is neither required nor recommended. Such agitation may negatively impact cell recovery, whereas static incubation ensures consistent labeling (per manufacturer's technical specifications for kit 130-090-101).

4. Add 1× binding buffer to the cell suspension to reach a minimum volume of 500 μL for MACS MS column separation.

5. Filter the cell suspension through a 40 μm cell strainer.

6. Place an MS column in the magnetic field of a suitable MACS Separator.

7. Prepare the column by rinsing with 0.5 mL of 1× binding buffer.

8. Apply the cell suspension onto the column reservoir and collect the flowthrough, which contains the unlabeled (live) cells, while the magnetically labeled dead cells are retained within the column.

9. Wash the column three times with 0.5 mL of 1× binding buffer each. Collect the unlabeled cells that pass through and combine with the flowthrough from step D8.

10. Centrifuge the combined effluents at 300× g for 10 min at 4 °C.

11. Wash the cells three times with 1 mL of washing buffer each.

12. Resuspend the cell pellet in 200 μL of washing buffer and count the cells using Trypan Blue by Countess^® II Cell Counter.

13. (Optional) If the live cell fraction is less than 90%, repeat the dead cell removal procedure as described in steps D2–11 with a new column.

Pause point: The experimental phase concludes here. All subsequent steps involve computational analysis of the acquired data.

Note: See General note 5 for the integration of biological replicates.

E. GEM generation and library construction

1. Dilute each sample from step C12 in washing buffer to a final concentration of 1 × 10⁶ cells/mL (1,000 cells/μL).

2. Combine 200 μL of labeled cells from each sample in a tube and mix well for gel beads-in-emulsion (GEMs) generation.

3. Perform GEM generation and construction of the 5′ gene expression and cell hashing libraries according to the Chromium Next GEM Single Cell 5’ Reagent kits v2 (Dual Index) User Guide with Feature Barcode technology for Cell Surface Protein & Immune Receptor Mapping (10× Genomics, CG000330 Rev A).

4. Perform sequencing of the libraries according to the guidelines in the user guide.

Data analysis

Following library preparation, the gene expression and cell hashing libraries were sequenced on an Illumina NovaSeq S4 platform using paired-end reads, aiming for a depth of 250 million and 50 million reads, respectively. Raw sequencing data were processed using the 10× Genomics Cell Ranger pipeline (v7.0) to perform demultiplexing, read alignment, and cellular barcode/UMI quantification. The resulting feature-barcode matrices were then imported into the Seurat R package (v5.0) for downstream analysis.

Initial quality control was performed to exclude low-quality cells based on a minimum threshold of 500 UMIs and 200 detected features (genes) per cell. Cells with a mitochondrial gene content exceeding 12% were filtered out to remove apoptotic or damaged cells. After batch effect correction, the data underwent dimensionality reduction and unsupervised clustering. Samples were demultiplexed on biotin-streptavidin hashtag barcodes (Figure 1). Cell types were annotated based on the expression of canonical markers (Figure 2). Differential gene expression was assessed to identify differentially expressed genes (DEGs) at both the cluster and sample levels. Finally, these DEGs were analyzed using Ingenuity Pathway Analysis (IPA, QIAGEN) to identify significantly enriched biological pathways and functional networks (Figure 3).

Figure 1. Validation of custom cell hashing and sample demultiplexing in FVB/N mice. (A) UMAP illustrating the specific signal intensity of each biotin-streptavidin hashtag barcode across the four experimental groups: WT-Ctrl, WT-LPS, mKO-Ctrl, and mKO-LPS. (B) UMAP visualization of the barcode clusters, demonstrating distinct separation of the four pooled samples and minimal cross-sample contamination, confirming high demultiplexing accuracy.

Figure 2. Cellular landscape and clustering of the FVB/N lung with and without lipopolysaccharide (LPS) exposure. (A) Integrated UMAP embedding of all barcode-certified cells from all four experimental groups (WT-Ctrl, WT-LPS, mKO-Ctrl, and mKO-LPS), colored by identified cell types. (B) Split-UMAP projections of the four experimental groups demonstrating consistent cell-type representation and shifts in cell population density (e.g., neutrophil recruitment) following LPS instillation in both WT and mKO strains.


Figure 3. Upstream regulator analysis validating the biological response to lipopolysaccharide (LPS) administration . (A) Ingenuity pathway analysis (IPA), identifying LPS and its downstream signaling hubs (e.g., TNF, IL1B) as the top-ranked upstream regulators based on the differential gene expression. (B) Radial regulation diagram, showing the predicted activation states and directional relationships between LPS and target molecules. Red/green nodes indicate observed up/down-regulation, while orange/blue lines represent predicted activation/inhibition connections. The attenuated gene activation in mKO mice reveals an impaired immune response following Pdlim2 deletion, demonstrating the protocol’s sensitivity in deconvoluting strain-specific mechanisms of action from a single multiplexed run.

Validation of protocol

This protocol has been used and validated in the following research article:

1. Gao et al. [7]. Myeloid PDLIM2 repression as a common mechanism of infection susceptibility in lung diseases. Front. Immunol. 2025;16:1669117 (Figures 3–5, Supplemental figures 1–2).

In summary, this protocol was functionally validated by resolving the lung microenvironments of FVB/N mice across four distinct experimental conditions. By utilizing a custom biotinylated antibody cocktail combined with streptavidin-conjugated oligos, we successfully demultiplexed over 7,000 cells with high signal-to-noise ratios and minimal doublet interference. The biological accuracy of the method was confirmed by three key metrics: (1) the identification of all major lung cell populations; (2) the robust detection of canonical LPS-responsive upstream regulators; and (3) the successful characterization of the compromised immune response as expected in the mKO-LPS group, demonstrating the protocol’s sensitivity in capturing strain-specific mechanistic differences.

General notes and troubleshooting

General notes

1. Sample pooling and dissociation: To minimize inter-animal biological variation, samples from the same treatment group may be pooled into a single gentleMACS C tube. This system accommodates volumes from 500 μL to 10 mL. When attaching the tube to the gentleMACS Dissociator, verify that the tissue is physically positioned within the rotor area to ensure uniform homogenization.

2. If a gentleMACS Octo Dissociator with Heaters is not available, manual incubation can be performed. Between the pre-set dissociation programs, remove the C tube and place it in a 37 °C water bath for 5–10 min with gentle agitation. Ensure the tube is properly re-attached and the tissue remains in the rotor area for each subsequent dissociation cycle.

3. Unique barcoding for multiplexing: In step C5, each biological sample must be labeled with a unique streptavidin-conjugated oligo barcode. Assigning distinct barcodes is the only way to computationally demultiplex individual samples from the shared pool following GEM generation and sequencing.

4. Scaling dead cell removal: During step D2, the volume of dead cell removal microbeads should be maintained as specified for any cell load up to 10⁷ cells. For samples exceeding 10⁷ cells, scale all reagent and total buffer volumes proportionally to prevent bead saturation and ensure optimal cell viability and recovery.

5. Integration of biological replicates: To ensure statistical rigor in larger studies, it is recommended that at least three biological replicates be used per experimental group. These biological replicates should be processed individually from the tissue dissociation step to the dead cell removal step (sections A–D). Each biological replicate (e.g., three different WT-LPS mice) should be labeled with a unique hashtag oligos (HTOs) during step C5. This enables the computational identification of “inter-individual” vs. “inter-group” variation in downstream analyses.

Troubleshooting

Problem 1: Low cell viability (<70%) after dissociation from step B20.

Possible causes: Excessive mechanical force or over-incubation in enzymes.

Solutions: Reduce the duration of the compression cycles in the gentleMACS program or ensure enzymes are kept on ice until the moment of use.

Problem 2: Low cell recovery after the MS column from step D12.

Possible causes: Cell clumping or column clogging.

Solutions: Ensure the cell suspension is filtered through a 40 μm strainer immediately before applying to the column (step D5). If clogging occurs, use a larger (LS) column.

Problem 3: High percentage of dead cells after MACS separation from step D13.

Possible cause: Reagent volume was scaled down for low cell counts.

Solution: Always use 100 μL of beads for any cell load up to 10⁷ cells. Do not scale down, as binding kinetics require a minimum concentration of colloidal beads.

Acknowledgments

Z.Q. and G.X. conceived and designed the study, led and contributed to all aspects of the analysis, and edited the manuscript. F.G. analyzed the data and wrote the manuscript. X.L. and F.S. performed and analyzed all the experimental assays. Y.X. contributed to mouse clone maintenance. The study is supported in part by the NIH National Institute of General Medical Sciences (NIGMS) grant R01 GM144890, National Cancer Institute (NCI) grant R01 CA258614, National Heart, Lung, and Blood Institute R01 HL177140, American Cancer Society (ACS) Research Scholar grant RSG-19-166-01-TBG, American Lung Association (ALA) Lung Cancer Discovery Award 821321, and Tobacco Related-Disease Research Program (TRDRP) Research Award T33IR6461. We also thank other team members for their critical reading and edits.

Competing interests

The authors declare that they have no competing interests.

Ethical considerations

All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh and the University of Southern California and carried out in accordance with NIH guidelines on animal care.

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