Parallel measurement of transcriptomes and proteomes from same single cells using nanodroplet splitting

Reagents and chemicals

Deionized water (18.2 MΩ) was purified using a Barnstead Nanopure Infinity system (Los Angeles, CA, USA). n-dodecyl-β-D-maltoside (DDM), CDK1 inhibitor RO-3306, iodoacetamide (IAA), ammonium bicarbonate (ABC), and formic acid (FA) were obtained from Sigma (St. Louis, MO, USA). Nuclease-free water (not DEPC-treated), mass spectrometry grade Trypsin Platinum (Promega, Madison, WI, USA) and Lys-C (Wako, Japan) were reconstituted in 50 mM ABC before usage. Dithiothreitol (DTT, No-Weigh format), acetonitrile (ACN) with 0.1% FA, and water with 0.1% FA (MS grade) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). SMART-Seq V4 Plus kit (Cat# R400753) was purchased from Takara Bio USA.

Design, fabrication, and assembly of the nanoSPLITS chips

The nanoSPLITS chips were fabricated using standard photolithography, wet etching, and silanization as described previously²². Two different chips were designed and used in this study. Both contained 48 (4 × 12) nanowells with a well diameter of 1.2 mm. The inter-well distance for the first chip was 2.5 mm while the second was 4.5 mm. Chip fabrication utilized a 25 mm × 75 mm glass slide pre-coated with chromium and photoresist (Telic Company, Valencia, USA). After photoresist exposure, development, and chromium etching (Transene), select areas of the chip were protected using Kapton tape before etching to a depth of ~5 µm with buffered hydrofluoric acid. The freshly etched slide was dried by heating it at 120 °C for 1 h and then treated with oxygen plasma for 3 min (AP-300, Nordson March, Concord, USA). 2% (v/v) heptadecafluoro-1,1,2,2-tetrahydrodecyl-dimethylchlorosilane (PFDS, Gelest, Germany) in 2,2,4-trimethylpentane was applied onto the chip surface and incubated for 30 min to allow for silanization. The remaining chromium covering the wells was removed with etchant, leaving elevated hydrophilic nanowells surrounded by a hydrophobic background. To prevent retention of mRNA via interaction with free silanols on the hydrophilic surface of the nanowells, freshly etched chips were exposed to chlorotrimethylsilane under vacuum overnight to passivate the glass surface. A glass frame was epoxied to a standard glass cover slide so that it could be easily removed from the 2.5 mm inter-well distance chips for droplet splitting. For the 4.5 mm inter-well distance chips, PEEK chip covers were machined to fit the chip. Chips were wrapped in parafilm and aluminum foil for long-term storage and intermediate steps during sample preparation.

Cell culture

Two murine cell lines (NAL1A clone C1C10 is referred to as C10 and is a non-transformed alveolar type II epithelial cell line derived from normal BALB/c mouse lungs; SVEC4-10, an endothelial cell line derived from axillary lymph node vessels) were cultured at 37 °C and 5% CO₂ in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum and 1× penicillin-streptomycin (Sigma, St. Louis, MO, USA). For the RO-3306 treatment, C10 cells were seeded at 200,000 cells per dish and incubated overnight. Treated cells were cultured with 10 µM RO-3306 for 36 h before harvesting. Control C10 cells were cultured similarly with vehicle (DMSO). The cultured cell lines were collected in a 15 ml tube and centrifuged at 1000 × g for 3 min to remove the medium. Cell pellets were washed three times by PBS, then counted to obtain cell concentration. PBS was then added to achieve a concentration of ~200 × 10⁶ cells/mL. Immediately before cell sorting, the cell-containing PBS solution was passed through a 40 µm cell strainer (Falcon™ Round-Bottom Polystyrene Test Tubes with Cell Strainer Snap Cap, FisherScientific) to remove aggregated cells.

Human islets (1000−3000 islet equivalents) were obtained from Prodo Laboratories. Islets were immediately cultured in PIM(S) media in a humidified incubator at 37 °C and 5% CO₂ after being received. After 24 h in PIM(S) media, islets cells were collected and centrifuged at 100 x g for 5 min and resuspended in TrypLE^TM for cell dissociation by incubating in a 37 °C water bath for 15 to 30 min along with gentle pipetting until islets were visibly dispersed and no clumps of cells remained. The dissociated islet cells were centrifuge at 300 x g for 5 min; the supernatant was removed, and cells were resuspended in PBS. The dissociated cells were stained with 0.1 µM Calcein AM and 15 µM propidium iodide for cell viability prior to cell sorting with the cellenONE.

nanoSPLITS buffer optimization experiment

Using nuclease-free water (Thermo Fisher Scientific, cat# 4387936), several 10 mM Tris pH 8 test buffers were created containing 0.1% DDM and/or 1 x RNase inhibitor. 10 µL of each buffer was added to four wells within a 96 well plate. 20 C10 cells were then sorted into each well before snap freezing with liquid nitrogen. Immediately after thawing and centrifugation at 2500 g, 5 µL from each well was transferred to a separate 96 well PCR plate containing 7.5 µL of 3’ SMART-Seq CDS primer II before heating 70 °C for 3 min. 7.5 µL of RT mix (4 µL 5x ultra low first-strand buffer, 1 µL 48 µM SMART-Seq V4 oligonucleotide, 0.5 µL 40 units/µL RNase inhibitor, 2 µL SMARTScribe II reverse transcriptase) was then added before incubation at 42 °C for 90 min and 70 °C for 10 min. 30 µL of PCR master mix (25 µL SeqAmp PCR buffer, 1 µL PCR primer II A, 3 µL water, 1 µL SeqAmp DNA polymerase) was then added to each tube before performing 18 cycles of PCR (98 °C for 10 s, 65 °C for 30 s, 68 °C for 3 min). Isolation of cDNA was performed with Ampure XP beads with 80% ethanol washes. cDNA concentration and quality were determined with a Qubit fluorometer and Agilent fragment analyzer before next generation-sequencing, respectively.

The remaining 5 µL was retained and processed for label free proteomic analysis. Briefly, 5 µL of extraction buffer containing DTT and DDM was added to cell lysate to bring each sample to a final concentration of 1 mM DTT and 0.1% DDM before incubation at 60 °C for 1 h. 2 µL of 12 mM IAA was then added for a final concentration of 2 mM IAA before a 30 min incubation at 37 °C. 2 µL of 2.5 ng/µL Lys-C and 10 ng/µL of trypsin was added before incubation at 37 °C for 10 h. Enzymatic digestion was quenched by adding formic acid to a concentration of 1% before drying samples under vacuum. Peptides were reconstituted in 3 µL 5% acetonitrile 0.1% FA and transferred to a polypropylene microPOTS for proteomic analysis.

Fluorescein nanoSPLITS experiment

To the fluorescein-containing nanoSPLITS chip, 200 nL 0.01% 5,6-carboxyfluorescein solution containing 0.1% DDM was dispensed onto each well. For the PBS-containing chip, 250 nL PBS solution containing 0.1% DDM was dispensed onto each well. A slide cover was placed on both chips before wrapping them tightly in aluminum foil and placing them on ice to prevent evaporation until imaging. For imaging, both chips were placed on a chilled aluminum slide-holder and immediately imaged with brightfield light, followed by a Cy2 spectral filter using an AlphaImager FluorChemQ. Following imaging of the unsplit chips, two 2 cm² of 1/32” thick polyethylene foam was placed on one chip. The upper-chip was slowly lowered onto the bottom-chip, while carefully aligning the wells on both chips. Once the upper-chip was sitting on the separating foam, equal pressure was applied on the sides of the chip so that the droplets from both chips merged. Pressure was held for 15 seconds before releasing. The droplets were merged twice more following this process. The post-split chips were immediately placed back in the imager and final images were acquired. Quantification of droplet splitting was performed with the “Fiji” distribution of Image J (version 1.5). Briefly, Cy2 emission images were converted to grayscale. Regions of interest were selected (chip wells) and analyzed using the standard particle analysis in ImageJ. Each region of interest produced an average pixel intensity that was normalized by droplet area before using for quantification.

CellenONE cell sorting and nanoSPLITS workflow

Before cell sorting, all nanoSPLITS chips were prepared by the addition of 200 nL hypotonic solution consisting of 0.1% DDM in 10 mM Tris, pH 8 to each nanowell. A cellenONE instrument equipped with a glass piezo capillary (P-20-CM) for dispensing and aspiration was utilized for single-cell isolation. Sorting parameters included a pulse length of 50 µs, a nozzle voltage of 80 V, a frequency of 500 Hz, a LED delay of 200 µs, and a LED pulse of 3 µs. The slide stage was operated at dew-point control mode to reduce droplet evaporation. Cells were isolated based on their size, circularity, and elongation in order to exclude apoptotic cells, doublets, or cell debris. For C10 cells, this corresponded to 25 to 40 µm in diameter, maximum circularity of 1.15, and maximum elongation of 2, while SVEC cells were 24 to 32 µm in diameter, maximum circularity of 1.15, and maximum elongation of 2. All cells were sorted based on bright field images in real time. The pooled C10 experiment had 11, 3, and 1 C10 cells sorted into each nanowell on a single 2.5 mm inter-well distance chip. For the SVEC and C10 comparison experiment, a single 48 well chip with 4.5 mm inter-well distance was used for each cell type and had a single cell sorted into each well. To perform the transferring identifications based on FAIMS filtering (TIFF) methodology for scProteomics²³, a library chip was also prepared containing 20 cells per nanowell, with each cell type sorted separately on the same chip to reduce technical variation. After sorting, all chips were wrapped in parafilm and aluminum foil before being snap-frozen and stored at −80 °C, which may partially serve to induce cell lysis with a single freeze-thaw. All associated settings, single-cell images, and metadata can be accessed at nanoSPLITS GitHub repository [https://github.com/Cajun-data/nanoSPLITS]⁵⁴.

To accomplish splitting of the cell lysate, chips were first allowed to thaw briefly on ice. For each split, a complementary chip was prepared that contained the same 200 nL of 0.1% DDM in 10 mM Tris, pH 8 on each nanowell. The bottom chip containing the cell lysate was placed on an aluminum chip holder that was pre-cooled to 4 °C within a PCR workstation (AirClean Systems AC600). Precut 1/32” thick polyurethane foam was placed around wells on the exterior of this bottom chip while the top chip was slowly lowered onto the polyurethane foam (Supplementary Movie 1). Wells were manually aligned for each chip before manual pressure was applied equally across the chip to merge the droplets for each chip. Pressure was held for 15 s before releasing. The droplets were merged twice more following this process. For consistency, the top chip was used for scRNAseq in all experiments while the bottom chip that initially contained the cell lysate was utilized in scProteomics (apart from the data generated for Supplementary Fig. 3). After merging, the top chip was immediately transferred into a 96-well or 384-well UV-treated plate containing RT-PCR reagents. For the pooled C10 (11, 3, and 1 cell) experiment, the transfer was performed by adding 1 µL of RT-PCR buffer to each nanowell before withdrawing the entire volume and adding it to a 96-well plate. For all other nanoSPLITS experiments, the transfer was accomplished by laying the 4.5 mm inter-well distance chip onto a 384-well plate containing wells with the RT-PCR mix, sealed with a PCR plate seal, and then centrifuged at 3500 x g for 1 min.

Sample preparation and LC-MS/MS analysis for scProteomics

All post-split chips were first allowed to dry out before placing them into the humidified nanoPOTS platform for sample processing. Protein extraction was accomplished by dispensing 150 nL of extraction buffer containing 50 mM ABC, 0.1% DDM, 0.3 x diluted PBS, and 2 mM DTT and incubating the chip at 50 °C for 90 min. Denatured and reduced proteins were alkylated through the addition of 50 nL 15 mM IAA before incubation for 30 min in darkness at room temperature. Alkylated proteins were then digested by adding 50 nL 50 mM ABC with 0.1 ng/nL of Lys-C and 0.4 ng/nL of trypsin and incubating at 37 °C overnight. The digestion reaction was then quenched by adding 50 nL of 5% formic acid before drying the chip under vacuum at room temperature. All chips were stored in a −20 °C until LC-MS analysis.

We employed the in-house assembled nanoPOTS autosampler for LC-MS analysis. The autosampler contains a custom packed SPE column (100 μm i.d., 4 cm, 5 μm particle size, 300 Å pore size C18 material, Phenomenex) and analytical LC column (50 μm i.d., 25 cm long, 1.7 μm particle size, 190 Å pore size C18 material, Waters) with a self-pack picofrit (cat. no. PF360-50-10-N-5, New Objective, Littleton, MA). The analytical column was heated to 50 °C using AgileSleeve column heater (Analytical Sales and services, Inc., Flanders, NJ). Briefly, samples were dissolved with Buffer A (0.1% formic acid in water) on the chip, then trapped on the SPE column for 5 min. After washing the peptides, samples were eluted at 100 nL/min and separated using a 60 min gradient from 8% to 35% Buffer B (0.1% formic acid in acetonitrile).

An Orbitrap Eclipse Tribrid MS (ThermoFisher Scientific) with FAIMS, operated in data-dependent acquisition mode, was used for all analyses. Source settings included a spray voltage of 2400 V, ion transfer tube temperature of 200 °C, and carrier gas flow of 4.6 L/min. For TIFF method²³ samples, ionized peptides were fractionated by the FAIMS interface using internal compensation voltage stepping (−45, −60, and −75 V) with a total cycle time of 0.8 s per compensation voltage. Fractionated ions within a mass range 350–1600 m/z were acquired at 120,000 resolution with a max injection time of 500 ms, AGC target of 1E6, RF lens of 30%. Tandem mass spectra were collected in the ion trap with an AGC target of 20,000, a “rapid” ion trap scan rate, an isolation window of 1.4 m/z, a maximum injection time of 120 ms, and a HCD collision energy of 30%. For TIFF, a single library sample was collected at each compensation voltage (−45, −60, and −75 V) using independent LC-MS runs with slight modifications to the above method where cycle time was increased to 2 s and maximum injection time was set to 118 ms. Precursor ions with a minimum intensity of 1E4 were selected for fragmentation by 30% HCD and scanned in an ion trap with an AGC of 2E4 and an IT of 150 ms. Precursor ions with intensities > 1E4 were fragmented by 30% HCD and scanned with an AGC of 2E4 and an IT of 254 ms.

RT-PCR, sequencing, and read mapping for scRNAseq

Following the transfer of samples into a 384-well plate containing RT-PCR buffer with 3’ SMART-Seq CDS Primer IIA (SMART-Seq® v4 PLUS Kit, cat# R400753), the samples were immediately denatured at 72 °C for 3 min and chilled on ice for at least 2 min. Full length cDNA was generated by adding RT mix to each tube and incubating at 42 °C for 90 min; followed by heat inactivation at 70 °C for 10 min. 18 cycles of cDNA amplification were done to generate enough cDNA for template library according to SMART-Seq® v4 PLUS Kit instruction. The SMART-Seq Library Prep Kit and Unique Dual Index Kit (cat# R400745) were used to generate barcoded template library for sequencing. Single-read sequencing of the cDNA libraries with a read length of 150 was performed on NextSeq 550 Sequencing System using NextSeq 500/550 High Output v2 kit (150 cycles, cat#20024907). Data quality was assessed with FastQC and read-trimming was conducted using BBDuk. Reads were aligned to the mouse genome (Genome Reference Consortium Mouse Build 39) or human genome (Human genome assembly GRCh38) using STAR. BAM file outputs were mapped to genes using ‘featureCounts‘ as part of the Subread package with default settings or using the -O, -M and –fraction settings to allow genes with overlapping features to be counted.

Database searching and data analysis

All proteomic data raw files were processed by FragPipe⁵⁵ (version 17.1 for C10 and SVEC cells, version 18.0 for G2/M arrested C10 cells, and version 20.0 for human islet cells) and searched against the Mus musculus or Homo sapiens UniProt protein sequence database with decoy sequences (UP000000589 containing 17,201 forward entries, accessed 12/21 and UP000005640 containing 20,596 forward entries, accessed 05/23). Search settings included a precursor mass tolerance of +/− 20 ppm, fragment mass tolerance of +/− 0.5 Da, deisotoping, strict trypsin as the enzyme (with allowance for N-terminal semi-tryptic peptides for the human pancreatic islet cell experiment), carbamidomethylation as a fixed modification, and several variable modifications, including oxidation of methionine, N-terminal acetylation, S/Y/T phosphorylation (for CDK1-inhibition experiment), and N-terminal pyro-glutamate formation at Q/E (for human pancreatic islet cell experiment). Protein and peptide identifications were filtered to a false discovery rate of less than 0.01 within FragPipe. For the TIFF methodology, IonQuant MBR and MaxLFQ were set to “TRUE” and library MS datasets were assigned as such during the data import step. An MBR FDR of 0.05 at ion level was used to reduce false matching and normalization of samples was not applied. FragPipe result files were then imported into RStudio (Build 461) for downstream analysis in the R environment (version 4.1.3). For the pooled C10 samples, median normalization was performed with the “median_normalization” function from the proDA R package separately for each level of input (11, 3, and 1 cells) in order to avoid distorting the expected feature intensities. With all other experiments, median normalization was performed across all single-cells. With regards to quality control filtering of scProteomics and scRNAseq data, cells with total identifications of more than 2 standard deviations reduced from the average within their respective modality were removed from analysis. For scRNAseq data, a minimum of 5 read counts were used to filter genes with low abundance for all experiments with C10 and SVEC cells. In cases where complete data was needed, imputation was performed with k-nearest neighbors imputation (n = 10 neighbors) with < 60% missing values.

For the pancreatic islet experiment, batch correction was performed with the ComBat.NA function from the MSnSet.utils R package. Sparse partial least squares discriminant analysis (spls-DA) was utilized based on previously described algorithm⁵⁶. From the scProteomic data, common contaminants were removed, and cells were filtered to have at least 3000 peptide identifications. Three cell types were used to create different classes: alpha, beta, and delta (the remaining cells were considered as an alternative class).10-fold cross-validation was used to evaluate the model’s predictive ability and balanced accuracy was used to assess sensitivity/specificity. GO analysis was performed with the gprofiler2 R package and web application. All raw p-values determined through statistical testing were corrected for multiple hypothesis testing (where relevant) using the Benjamini-Hochberg procedure. All experiments were assessed with standardized quality control metrics (e.g., sensitivity and data completeness) as recommended by Vanderaa and Gatto (Supplementary Fig. 20)⁵⁷.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41467-024-54099-z