Fig. 3: scCLEAN improves immune cell type characterization with single-cell MAS-Seq.

a Schematic illustrating experimental workflow to incorporate scCLEAN into the MAS-Seq method. An amplified barcoded-cDNA pool generated with the 10X Genomics Chromium Controller was split and processed with MAS-Seq alone or in combination with scCLEAN (b) Sankey diagram as in Fig. 2a, except showing the flow from the MAS-seq method alone (left) to MAS-Seq with scCLEAN (right). c Transcripts aligning to the 255 gene panel were masked and the subsequent total number of identified transcript counts were binned into four categories via SQANTI3: full splice match (FSM), incomplete splice match (ISM), novel-in-catalog (NIC), and novel-not-in-catalog (NNIC). Differences in transcript quantities between 10x-V3 (blue) and scCLEAN (yellow) for each category were measured using a two-tailed t-test (n = 3; ns = p > 0.05, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001). Error bars represent ±SEM. d Transcripts aligning to the 255 gene panel were masked and (left) histograms of the shift in distributions of UMIs per cell and genes per cell comparing 10x-V3 and scCLEAN are shown. (right) Violin plots of UMIs per cell and genes per cell. Each replicate (n = 3) is shown. Significance was quantified using a two-tailed Wilcoxon rank-sum test with Bonferroni correction (**** = p ≤ 0.0001). e UMAP plots representing the query-to-reference labels using the Azimuth PBMC reference. Comparison between 10x-V3 (left), scCLEAN (middle), and the cell types uniquely identified within scCLEAN (right). f Cells labeled as cDC1 within the scCLEAN condition were selected and treated as bulk. Track plots indicate read coverage over top 3 cDC1 markers (COX5A, BTLA, and ENPP1) comparing 10x-V3 (red), scCLEAN (blue), and overlap (purple). Ensembl transcript IDs are annotated below. Box plots depict the minimum, first quartile (Q1), median, third quartile (Q3), and maximum values for each group. Whiskers extend to the minimum and maximum values within 1.5 × IQR from the quartiles. Detailed statistical values are available in Supplementary Data 4.