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Figure S1. Single cell DNA-seq of singly-edited Ba/F3 cell lines. QC metrics of single-cell DNA-seq analysis by Tapestri including number of sequencing reads and targeted amplicon coverage, analysis of percent modified reads across on and off-targets and zygosity analysis of on-targets. Figure S2. Single cell qPCR-based detection of sgRNAs. Analysis of number and co-occurrence of sgRNAs in single Ba/F3 cells, as assessed via qPCR-based detection. (DOCX 689 kb)

Standard RNA probe curves using lysed and diluted MCF7 cells. Two-fold dilutions of population lysate were backloaded into the C1 IFC and processed according to the PEA/STA protocol. Shown here are the STA measurements with the y-axis values representing ∆Ct values from lysis buffer alone or a threshold value of 24 if undetected in pure lysate. Certain assays (e.g. ACTB_R) display a “hook” effect, as seen in Additional files 2 and 3: Figures S1 and S2, potentially due to reagent saturation. Each data point plotted is the average of eight separate capture sites in the C1 IFC with error bars showing the standard error of the mean. Gray (green) dashes show the level above which the probability for a detection event being real is p = 0.01 (0.05). Points used for fitting the red trend line are colored blue, the background plot color indicates which treatment cells were taken from (0 hr = purple, 24 hr = green, 48 hr = blue). Genes are categorized as (a) usable or (b) unusable. (PDF 1087 kb)

Standard protein probe curves using lysed and diluted MCF7 cells. Two-fold dilutions of population lysate were backloaded into the C1 IFC and processed according to the PEA/STA protocol. Shown here are the PEA measurements with the y-axis values representing ∆Ct, which are calculated as the signal over a lysis buffer only control. Certain assays (e.g. EIF4EBP1_P) display a “hook” effect. The effect occurs when the concentration of a target protein exceeds a threshold beyond which PEA probes begin to occupy separate target molecules as opposed to the same one. This results in a reduction of signal due to a reduction in the number of proximal events. Each data point plotted is the average of eight separate capture sites in the C1 IFC with error bars showing the standard error of the mean. Gray (green) dashes show the level above which the probability for a detection event being real is p = 0.01 (0.05). Points used for fitting the red trend line are colored blue, the background plot color indicates which treatment cells were taken from (0 hr = purple, 24 hr = green, 48 hr = blue). Probes are categorized as (a) usable or (b) unusable. (PDF 557 kb)

Standard RNA probe curves using ERCC Spike-Ins. Two-fold dilutions of ERCC Spike-Ins were backloaded into the C1 IFC and processed according to the PEA/STA protocol. Shown here are the STA measurements with the y-axis values representing â Ct values from only lysis buffer or a threshold value of 24 if undetected in lysis buffer alone. Plots are ordered by decreasing concentration in the ERCC mix with bad fits arising around ERCC 14 (which corresponds to ~121 molecules loaded into the top dilution, Additional file 1: Table S10 and S11). Each data point plotted is the average of eight separate capture sites in the C1 IFC with error bars showing the standard error of the mean. Gray (green) dashes show the level above which the probability for a detection event being real is pâ =â 0.01 (0.05). Points used for fitting the red trend line are colored blue. (PDF 1135 kb)

Change in average, standard deviation, and frequency of expression. Density traces (each with their own arbitrary units) for change in mean, standard deviation, and frequency of expression are shown for genes quantified as both RNA (a) and protein (b). Each row depicts a time point transition (24 – 0 hr, 48 – 24 hr, 48 – 0 hr for the top, middle, and bottom rows, respectively) for every gene with at least two cells above detection in every time point for both RNA and protein (19 genes). The ticks display individual measurements from each time point transition. (PDF 508 kb)

Standard RNA probe curves using ERCC Spike-Ins. Two-fold dilutions of ERCC Spike-Ins were backloaded into the C1 IFC and processed according to the PEA/STA protocol. Shown here are the STA measurements with the y-axis values representing â Ct values from only lysis buffer or a threshold value of 24 if undetected in lysis buffer alone. Plots are ordered by decreasing concentration in the ERCC mix with bad fits arising around ERCC 14 (which corresponds to ~121 molecules loaded into the top dilution, Additional file 1: Table S10 and S11). Each data point plotted is the average of eight separate capture sites in the C1 IFC with error bars showing the standard error of the mean. Gray (green) dashes show the level above which the probability for a detection event being real is pâ =â 0.01 (0.05). Points used for fitting the red trend line are colored blue. (PDF 1135 kb)

Cell capture and GFP+/â signal of cells on the Fluidigm C1. (PDF 55 kb)

Correlation between protein and RNA: MET. Presented are the correlations of the various MET STAs with MET protein (by PEA) across time points (0 hr = purple, 24 hr = green, 48 hr = blue). We observe that the strong increase in correlation after stimulation is most noted in MET.5_R (spliced short isoform) and MET.6_R (exon 10), with the other STAs showing a modest increase in correlation at 24Â hr that becomes negative by 48Â hr. (PDF 122 kb)

Analyzing gene trajectories through a time course of PMA stimulation. Genes quantified as both RNA and protein were made binary for each value based on detection or lack thereof. The vector of the proportion of cells in the four quadrants of detected and undetected for RNA and protein across the three stimulation time points were clustered using Spearman correlation. Clusters are divided using a distance metric of 0.75; representative trajectories through time (depicted by color 0 hr = purple, 24 hr = green, 48 hr = blue) are shown for the genes evaluated in situ with the bottom left quadrant representing undetected protein and undetected RNA, the bottom right quadrant representing undetected protein and detected RNA, the top left quadrant representing detected protein and undetected RNA, and the top right quadrant representing detected protein and detected RNA. The size of the dots corresponds to the fraction of cells in that quadrant. (PDF 179 kb)

PCA separation of the various time points (0 hr = purple, 24 hr = green, 48 hr = blue). a A PCA over all quantitative protein targets and the corresponding ROC curves (b) for all three time points generated from random forest decision categorization with AUC of 0.98, 0.94, and 0.86 for 0 hr, 24 hr, and 48 hr, respectively. c A PCA over all quantitative RNA targets and the corresponding ROC curves (d) for all three time points generated from random forest decision categorization with AUC of 0.81, 0.80, and 0.57 for 0 hr, 24 hr, and 48 hr, respectively. e A PCA over all quantitative protein and RNA targets and the corresponding ROC curves (f) for all three time points generated from random forest decision categorization with AUC of 0.99, 0.94, and 0.84 for 0 hr, 24 hr, and 48 hr, respectively. For a, c, e, axis labels indicate which PC was used and what percent variance it explains. (PDF 269 kb)

Coefficient of variation colored by correlation. Genes quantified as both RNA and protein are plotted based on their standard deviation (y-axis) and mean (x-axis) on the left and right hand sides, respectively. The plots in the top, middle, and bottom rows are done at the 0Â hr (purple), 24Â hr (green), and 48Â hr (blue) time points, respectively. Dashed lines follow the standard deviation of a gene that has only ten cells with uniform expression and the remaining cells have undetectable levels (strong bimodality). (PDF 233 kb)

Additional benchmarking of a combined PEA/STA workflow: ICAM1. aâ c Validation of protein and RNA detection in single cells using a coupled PEA/STA script on the C1 throughout a PMA perturbation time course (0 hr = purple, 24 hr = green, 48 hr = blue). a RNA-FISH and IF staining of ICAM1 was performed to validate the C1-based, high-throughput RNA and protein measurements. Cyan (left) shows cell nuclei and boundaries, magenta ICAM1 protein (middle), and yellow ICAM1 RNA (right). Scale bars indicate 25 Îźm. b Qualitative agreement between the protein and RNA data obtained in situ and on the C1. Density distributions (each with their own arbitrary units) for ICAM1 RNA (left) and protein (right) obtained via qPCR (top) or in situ (bottom) staining. c Q-Q plots showing the range over which the PEA/STA measurements of ICAM1 track linearly with IF staining or in situ hybridization for the same. (PDF 107675 kb)

Additional benchmarking of a combined PEA/STA workflow: CASP8. aâ c Validation of protein and RNA detection in single cells using a coupled PEA/STA script on the C1 throughout a PMA perturbation time course (0 hr = purple, 24 hr = green, 48 hr = blue). a RNA-FISH and IF staining of CASP8 was performed to validate the C1-based, high-throughput RNA and protein measurements. Cyan (left) shows cell nuclei and boundaries, magenta CASP8 protein (middle), and yellow CASP8 RNA (right). Scale bars indicate 25 Îźm. b Qualitative agreement between the protein and RNA data obtained in situ and on the C1. Density distributions (each with their own arbitrary units) for CASP8 RNA (left) and protein (right) obtained via qPCR (top) or in situ (bottom) staining. c Q-Q plots showing the range over which the PEA/STA measurements of CASP8 track linearly with IF staining or in situ hybridization for the same. (PDF 124616 kb)

Additional benchmarking of a combined PEA/STA workflow: ICAM1. aâ c Validation of protein and RNA detection in single cells using a coupled PEA/STA script on the C1 throughout a PMA perturbation time course (0 hr = purple, 24 hr = green, 48 hr = blue). a RNA-FISH and IF staining of ICAM1 was performed to validate the C1-based, high-throughput RNA and protein measurements. Cyan (left) shows cell nuclei and boundaries, magenta ICAM1 protein (middle), and yellow ICAM1 RNA (right). Scale bars indicate 25 Îźm. b Qualitative agreement between the protein and RNA data obtained in situ and on the C1. Density distributions (each with their own arbitrary units) for ICAM1 RNA (left) and protein (right) obtained via qPCR (top) or in situ (bottom) staining. c Q-Q plots showing the range over which the PEA/STA measurements of ICAM1 track linearly with IF staining or in situ hybridization for the same. (PDF 107675 kb)

Spliced vs. unspliced MET transcript. Scatterplot of MET.5_R (spliced, short isoform) vs. MET.3_R (unspliced) with density curves plotted along the axes. Time point indicated by color: 0 hr = purple, 24 hr = green, 48 hr = blue. (PDF 227 kb)

In silico gating of samples by both discrete and continuous methods. a, b Heatmaps show protein or RNA targets (rows) significantly dysregulated when applying protein or RNA expression gates (columns). Cooler colors indicate a decrease in expressing cells by a Fisher’s exact test (a) or the distribution of expressing cells by a Mann–Whitney U test (b) while warmer colors indicate the inverse. c, d In addition to discrete gating, unsupervised analyses, such as a PCA over protein (c) or RNA levels (d), can also be annotated by additional cellular observations (RNA or protein data, respectively). c ESR1_R expression shows a strong Spearman's ρ of 0.60 with PC2 on a PCA computed from the protein expression. d AXIN1_P expression shows a strong negative ρ of −0.68 with PC1 on a PCA computed over RNA expression. (PDF 964 kb)

Controlling for sequencing depth. Similar results were obtained when subsampling to 300,000 reads. A, B Same as Fig. 1c, d with the equal subsampling. Câ G Same as Fig. 2 with the equal subsampling. (PDF 2999 kb)

Standard protein probe curves using recombinant proteins. Two-fold dilutions of recombinant proteins were backloaded into the C1 IFC and processed according to the PEA/STA protocol. Shown here are the PEA measurements, with the y-axis values representing â Ct values from only lysis buffer. Gray (green) dashes show the level above which the probability for a detection event being real is pâ =â 0.01 (0.05). Each data point plotted is the average of eight separate capture sites in the C1 IFC with error bars showing the standard error of the mean. Points used for fitting the red trend line are colored blue. Most probes evaluated with recombinants worked well (a) with the exception of CSF3R_P and TP53_P (b), whose lack of detection was also seen in the protein lysate dilutions (Additional file 3: Figure S2). (PDF 315 kb)

Optimization of the CEL-Seq protocol. A Number of genes obtained from ten replicates of 100Â pg RNA performed with each type of primer: the original primer, the original primer with the inclusion of UMI, and the shortened UMI primer. B Number of transcripts identified for the two primers containing a UMI. C Estimating the efficiency of CEL-Seq using UMIs and ERCC spike-ins. The efficiency is computed as the y-intercept. D Side-by-side comparison of column clean-up, bead clean-up, and two RTs relative to CEL-Seq with a UMI primer. E Side-by-side comparison of different second-strand synthesis enzymes. The MessageAmp II enzyme was the one used originally. (PDF 519 kb)

Analyzing population lysate dilution data. The Ct values observed for the two PEA standards: the extension control (a) and oligo reference (b). Across time points (0 hr = purple, 24 hr = green, 48 hr = blue) and cell dilutions, the deviations from the lysis buffer control are quite small except for the 42.5 and 21.25 cell equivalent dilutions (0.05 and 0.01 probability cutoffs from the background shown in green and gray, respectively). In these deviations are quite small except for the 42.5 and 21.25 cell equivalent dilutions. Therefore, those measurements are excluded from the dilution plots in Additional files 2, 3, and 4: Figures S1, S2, and S3. (PDF 143 kb)

Standard protein probe curves using recombinant proteins. Two-fold dilutions of recombinant proteins were backloaded into the C1 IFC and processed according to the PEA/STA protocol. Shown here are the PEA measurements, with the y-axis values representing â Ct values from only lysis buffer. Gray (green) dashes show the level above which the probability for a detection event being real is pâ =â 0.01 (0.05). Each data point plotted is the average of eight separate capture sites in the C1 IFC with error bars showing the standard error of the mean. Points used for fitting the red trend line are colored blue. Most probes evaluated with recombinants worked well (a) with the exception of CSF3R_P and TP53_P (b), whose lack of detection was also seen in the protein lysate dilutions (Additional file 3: Figure S2). (PDF 315 kb)

Additional benchmarking of a combined PEA/STA workflow: CASP8. aâ c Validation of protein and RNA detection in single cells using a coupled PEA/STA script on the C1 throughout a PMA perturbation time course (0 hr = purple, 24 hr = green, 48 hr = blue). a RNA-FISH and IF staining of CASP8 was performed to validate the C1-based, high-throughput RNA and protein measurements. Cyan (left) shows cell nuclei and boundaries, magenta CASP8 protein (middle), and yellow CASP8 RNA (right). Scale bars indicate 25 Îźm. b Qualitative agreement between the protein and RNA data obtained in situ and on the C1. Density distributions (each with their own arbitrary units) for CASP8 RNA (left) and protein (right) obtained via qPCR (top) or in situ (bottom) staining. c Q-Q plots showing the range over which the PEA/STA measurements of CASP8 track linearly with IF staining or in situ hybridization for the same. (PDF 124616 kb)

Standard protein probe curves using lysed and diluted MCF7 cells. Two-fold dilutions of population lysate were backloaded into the C1 IFC and processed according to the PEA/STA protocol. Shown here are the PEA measurements with the y-axis values representing ∆Ct, which are calculated as the signal over a lysis buffer only control. Certain assays (e.g. EIF4EBP1_P) display a “hook” effect. The effect occurs when the concentration of a target protein exceeds a threshold beyond which PEA probes begin to occupy separate target molecules as opposed to the same one. This results in a reduction of signal due to a reduction in the number of proximal events. Each data point plotted is the average of eight separate capture sites in the C1 IFC with error bars showing the standard error of the mean. Gray (green) dashes show the level above which the probability for a detection event being real is p = 0.01 (0.05). Points used for fitting the red trend line are colored blue, the background plot color indicates which treatment cells were taken from (0 hr = purple, 24 hr = green, 48 hr = blue). Probes are categorized as (a) usable or (b) unusable. (PDF 557 kb)

PCA separation of the various time points (0 hr = purple, 24 hr = green, 48 hr = blue). a A PCA over all quantitative protein targets and the corresponding ROC curves (b) for all three time points generated from random forest decision categorization with AUC of 0.98, 0.94, and 0.86 for 0 hr, 24 hr, and 48 hr, respectively. c A PCA over all quantitative RNA targets and the corresponding ROC curves (d) for all three time points generated from random forest decision categorization with AUC of 0.81, 0.80, and 0.57 for 0 hr, 24 hr, and 48 hr, respectively. e A PCA over all quantitative protein and RNA targets and the corresponding ROC curves (f) for all three time points generated from random forest decision categorization with AUC of 0.99, 0.94, and 0.84 for 0 hr, 24 hr, and 48 hr, respectively. For a, c, e, axis labels indicate which PC was used and what percent variance it explains. (PDF 269 kb)

Additional benchmarking of a combined PEA/STA workflow: BIRC5. aâ c Validation of protein and RNA detection in single cells using a coupled PEA/STA script on the C1 throughout a PMA perturbation time course (0 hr = purple, 24 hr = green, 48 hr = blue). a RNA-FISH and IF staining of BIRC5 was performed to validate the C1-based, high-throughput RNA and protein measurements. Cyan (left) shows cell nuclei and boundaries, magenta BIRC5 protein (middle), and yellow BIRC5 RNA (right). Scale bars indicate 25 Îźm. b Qualitative agreement between the protein and RNA data obtained in situ and on the C1. Density distributions (each with their own arbitrary units) for BIRC5 RNA (left) and protein (right) obtained via qPCR (top) or in situ (bottom) staining. c Q-Q plots showing the range over which the PEA/STA measurements of BIRC5 track linearly with IF staining or in situ hybridization for the same. (PDF 79799 kb)

Standard RNA probe curves using lysed and diluted MCF7 cells. Two-fold dilutions of population lysate were backloaded into the C1 IFC and processed according to the PEA/STA protocol. Shown here are the STA measurements with the y-axis values representing ∆Ct values from lysis buffer alone or a threshold value of 24 if undetected in pure lysate. Certain assays (e.g. ACTB_R) display a “hook” effect, as seen in Additional files 2 and 3: Figures S1 and S2, potentially due to reagent saturation. Each data point plotted is the average of eight separate capture sites in the C1 IFC with error bars showing the standard error of the mean. Gray (green) dashes show the level above which the probability for a detection event being real is p = 0.01 (0.05). Points used for fitting the red trend line are colored blue, the background plot color indicates which treatment cells were taken from (0 hr = purple, 24 hr = green, 48 hr = blue). Genes are categorized as (a) usable or (b) unusable. (PDF 1087 kb)

Additional benchmarking of a combined PEA/STA workflow: BIRC5. aâ c Validation of protein and RNA detection in single cells using a coupled PEA/STA script on the C1 throughout a PMA perturbation time course (0 hr = purple, 24 hr = green, 48 hr = blue). a RNA-FISH and IF staining of BIRC5 was performed to validate the C1-based, high-throughput RNA and protein measurements. Cyan (left) shows cell nuclei and boundaries, magenta BIRC5 protein (middle), and yellow BIRC5 RNA (right). Scale bars indicate 25 Îźm. b Qualitative agreement between the protein and RNA data obtained in situ and on the C1. Density distributions (each with their own arbitrary units) for BIRC5 RNA (left) and protein (right) obtained via qPCR (top) or in situ (bottom) staining. c Q-Q plots showing the range over which the PEA/STA measurements of BIRC5 track linearly with IF staining or in situ hybridization for the same. (PDF 79799 kb)

The sequences of the CEL-Seq primers. The 8 base barcodes are as previously published, the 6 base barcodes are listed in the detailed protocol. (PDF 43 kb)

The sequences of the CEL-Seq primers. The 8 base barcodes are as previously published, the 6 base barcodes are listed in the detailed protocol. (PDF 43 kb)

Table S1. Mass cytometry panel for analysis of synovial cells. Figure S1. Additional variables in synovial tissue enzymatic treatment (PDF 2239 kb)

Supplementary file 2. CEL-Seq in C1. The file includes the complete information for performing CEL-Seq on the Fluidigm C1 instrument. (DOCX 17 kb)

Table S1. Mass cytometry panel for analysis of synovial cells. Figure S1. Additional variables in synovial tissue enzymatic treatment (PDF 2239 kb)

Supplementary file 1. The CEL-Seq2 protocol. This file includes the detailed CEL-Seq2 protocol. (DOCX 279 kb)

Supplementary file 1. The CEL-Seq2 protocol. This file includes the detailed CEL-Seq2 protocol. (DOCX 279 kb)

Review history. (PDF 105 kb)

Table S1. Mass cytometry panel for analysis of synovial cells. Figure S1. Additional variables in synovial tissue enzymatic treatment (PDF 2239 kb)

Review history. (PDF 105 kb)