Journal: Nature Communications

Article Title: Single-cell exon deletion profiling reveals splicing events that shape gene expression and cell state dynamics

doi: 10.1038/s41467-026-68774-w

Figure Lengend Snippet: a Heatmap of pairwise correlations between log2 fold-change values of differentially expressed genes across gene knockout and exon deletion (_ex) perturbations identified by scCHyMErA-Seq. Distinct sub-clusters are annotated with Molecular Signatures Database (MSigDB) pathway terms. Adjusted p values for pathway enrichment are indicated; **** p < 0.0001, ** p < 0.01; one-sided Fisher’s exact test corrected for multiple hypothesis (Benjamini–Hochberg adjusted). Representative genes from each subcluster are listed on the right. b Heatmap depicting gene expression changes in replication-dependent histones across the indicated perturbed exons. All the genes that are commonly upregulated following LSM11 exon-3 and TAF5 exon-8 deletions are shown. NRF1 exon-7 deletion serves as a control. c RT-PCR analysis of TAF5 exon-8 (top) and LSM11 exon-3 (bottom) inclusion in RNA from HAP1 cells transduced with three independent intergenic hgRNA control sequences or three independent hgRNA sequences targeting TAF5 exon-8 or LSM11 exon-3 for deletion. Constructs compatible with the scCHyMErA-Seq system were used for exon deletion. d qRT-PCR analysis of polyadenylated histone gene transcripts following deletion of TAF5 exon-8 or LSM11 exon-3 using the three independent intergenic hgRNA controls or exon-targeting hgRNAs shown in Fig. 3c. Data represent mean ± SD from three biological replicates; ** p < 0.01, * p < 0.05, two-tailed unpaired t -test.

Article Snippet: Proteins were transferred to PVDF membranes (Immobilon-P, Millipore #IPVH00010) using a Mini Blot Module (Life Technologies) at 22 V for 60 min. Membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against NRF1 (1:2000; Proteintech #12482-1-AP), TAF5 (1:2000; Thermo Fisher Scientific #MA3-076), SRSF3 (1:1000; MBL Life Science #RN080PW), FLAG (1:2500; Sigma-Aldrich #F3165), GAPDH (1:2500; Proteintech #10494-1-AP), and β-tubulin (1:2500; Proteintech #10094-1-AP).

Techniques: Gene Knockout, Gene Expression, Control, Reverse Transcription Polymerase Chain Reaction, Transduction, Construct, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: Single-cell exon deletion profiling reveals splicing events that shape gene expression and cell state dynamics

doi: 10.1038/s41467-026-68774-w

Figure Lengend Snippet: a Schematic of NRF1 protein domains and exon structure. The protein diagram (top) highlights low-complexity regions (orange), the DNA-binding domain (blue), and the transcriptional activation domain (turquoise). The splicing schematic (middle) depicts the full-length (FL, top) and exon-7–deleted (ΔE7, bottom) mRNA isoforms. AlphaFold structural predictions , for the NRF1 full-length (top right) and Δexon-7 (bottom right) isoforms are shown, with the exon-7-encoded region highlighted in red. b Volcano plot of DESeq2 pseudobulk analysis comparing cells expressing NRF1 exon-7–targeting hgRNAs with intergenic controls. Significantly upregulated (red) and downregulated (blue) genes are indicated (adjusted p < 0.05 and |log2 fold-change|>0.5). p values were computed using DESeq2’s Wald test with Benjamini–Hochberg correction. c RT-PCR analysis of NRF1 exon-7 inclusion in RNA from HAP1 cells transduced with three independent intergenic control hgRNAs or three independent hgRNAs targeting NRF1 exon-7 for deletion using scCHyMErA-Seq-compatible constructs. Representative images from three independent experiments. d qRT-PCR validation of NRF1 exon-7-dependent gene expression changes using the hgRNAs shown in Fig. 4c. Data represent mean ± SD from three biological replicates; **** p < 0.0001, *** p < 0.001, ** p < 0.01; two-way ANOVA with Dunnett’s multiple comparisons test. e Western blot analysis of NRF1 isoforms in HEK293 Flp-In cells stably expressing doxycycline-inducible 3×FLAG-tagged NRF1 with exon-7 included (FL) or excluded (ΔE7). The ectopic isoforms carry silent mutations at the siRNA target site, conferring resistance to siNRF1. Cells were transfected with control (siNT) or NRF1-targeting (siNRF1) siRNAs. Blots were probed for NRF1, FLAG, and GAPDH (loading control). Representative images from three independent experiments. f RNA-Seq analysis showing Z-score normalized expression changes of differentially expressed genes in siNRF1-treated cells rescued with NRF1-FL or -∆E7 isoforms. g ChIP-Seq density profiles of NRF1-FL and -∆E7 isoform enrichment over input at transcription start sites (TSS) ± 5 kb flanking regions of the genes regulated by NRF1 exon-7. h siRNA screen of 60 splicing regulators identifying factors controlling NRF1 exon-7 inclusion. Data represent the mean ± SD from three biological replicates; **** p < 0.0001, ** p < 0.01; two-way ANOVA.

Article Snippet: Proteins were transferred to PVDF membranes (Immobilon-P, Millipore #IPVH00010) using a Mini Blot Module (Life Technologies) at 22 V for 60 min. Membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against NRF1 (1:2000; Proteintech #12482-1-AP), TAF5 (1:2000; Thermo Fisher Scientific #MA3-076), SRSF3 (1:1000; MBL Life Science #RN080PW), FLAG (1:2500; Sigma-Aldrich #F3165), GAPDH (1:2500; Proteintech #10494-1-AP), and β-tubulin (1:2500; Proteintech #10094-1-AP).

Techniques: Binding Assay, Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Transduction, Control, Construct, Quantitative RT-PCR, Biomarker Discovery, Gene Expression, Western Blot, Stable Transfection, Transfection, RNA Sequencing, ChIP-sequencing

Journal: Nature Communications

Article Title: Single-cell exon deletion profiling reveals splicing events that shape gene expression and cell state dynamics

doi: 10.1038/s41467-026-68774-w

Figure Lengend Snippet: a , c , e Venn diagrams showing the overlap of differentially expressed genes regulated by perturbation of NRF1 exon-7 ( a , c ) or TAF5 exon-8 ( e ). Overlaps are compared between our original scCHyMErA-Seq screen in HAP1 cells and either a small-scale replicate screen in HAP1 cells ( a ) or bulk RNA-Seq in HEK293 cells following isoform rescue experiments ( c , e ). Statistical significance was determined by two-sided Fisher’s exact test (**** p < 0.0001), and the corresponding odds ratios (OR) are indicated. b , d , f Scatterplots showing the Pearson correlation of log2 fold-changes for differentially expressed genes identified by scCHyMErA-Seq and either a replicate screen ( b ) or bulk RNA-Seq for NRF1 exon-7 ( d ) and TAF5 exon-8 ( f ) perturbations. Each point represents a gene; Two-sided Pearson correlation coefficients with corresponding significance values are indicated.

Article Snippet: Proteins were transferred to PVDF membranes (Immobilon-P, Millipore #IPVH00010) using a Mini Blot Module (Life Technologies) at 22 V for 60 min. Membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against NRF1 (1:2000; Proteintech #12482-1-AP), TAF5 (1:2000; Thermo Fisher Scientific #MA3-076), SRSF3 (1:1000; MBL Life Science #RN080PW), FLAG (1:2500; Sigma-Aldrich #F3165), GAPDH (1:2500; Proteintech #10494-1-AP), and β-tubulin (1:2500; Proteintech #10094-1-AP).

Techniques: RNA Sequencing

Journal: Nature Communications

Article Title: Single-cell exon deletion profiling reveals splicing events that shape gene expression and cell state dynamics

doi: 10.1038/s41467-026-68774-w

Figure Lengend Snippet: a Heatmap of pairwise correlations between log2 fold-change values of differentially expressed genes across gene knockout and exon deletion (_ex) perturbations identified by scCHyMErA-Seq. Distinct sub-clusters are annotated with Molecular Signatures Database (MSigDB) pathway terms. Adjusted p values for pathway enrichment are indicated; **** p < 0.0001, ** p < 0.01; one-sided Fisher’s exact test corrected for multiple hypothesis (Benjamini–Hochberg adjusted). Representative genes from each subcluster are listed on the right. b Heatmap depicting gene expression changes in replication-dependent histones across the indicated perturbed exons. All the genes that are commonly upregulated following LSM11 exon-3 and TAF5 exon-8 deletions are shown. NRF1 exon-7 deletion serves as a control. c RT-PCR analysis of TAF5 exon-8 (top) and LSM11 exon-3 (bottom) inclusion in RNA from HAP1 cells transduced with three independent intergenic hgRNA control sequences or three independent hgRNA sequences targeting TAF5 exon-8 or LSM11 exon-3 for deletion. Constructs compatible with the scCHyMErA-Seq system were used for exon deletion. d qRT-PCR analysis of polyadenylated histone gene transcripts following deletion of TAF5 exon-8 or LSM11 exon-3 using the three independent intergenic hgRNA controls or exon-targeting hgRNAs shown in Fig. 3c. Data represent mean ± SD from three biological replicates; ** p < 0.01, * p < 0.05, two-tailed unpaired t -test.

Article Snippet: After fixation, cells were permeabilized in 0.2% Triton X-100 (Sigma-Aldrich #T8787) for 10 min, followed by blocking in 5% BSA (Sigma-Aldrich #A9647) for 1 h. Primary antibodies against NRF1 (1:500; Proteintech #12482-1-AP) and FLAG (1:500; Sigma-Aldrich #F3165) were applied for 1 h at room temperature in DPBS containing 5% BSA.

Techniques: Gene Knockout, Gene Expression, Control, Reverse Transcription Polymerase Chain Reaction, Transduction, Construct, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: Single-cell exon deletion profiling reveals splicing events that shape gene expression and cell state dynamics

doi: 10.1038/s41467-026-68774-w

Figure Lengend Snippet: a Schematic of NRF1 protein domains and exon structure. The protein diagram (top) highlights low-complexity regions (orange), the DNA-binding domain (blue), and the transcriptional activation domain (turquoise). The splicing schematic (middle) depicts the full-length (FL, top) and exon-7–deleted (ΔE7, bottom) mRNA isoforms. AlphaFold structural predictions , for the NRF1 full-length (top right) and Δexon-7 (bottom right) isoforms are shown, with the exon-7-encoded region highlighted in red. b Volcano plot of DESeq2 pseudobulk analysis comparing cells expressing NRF1 exon-7–targeting hgRNAs with intergenic controls. Significantly upregulated (red) and downregulated (blue) genes are indicated (adjusted p < 0.05 and |log2 fold-change|>0.5). p values were computed using DESeq2’s Wald test with Benjamini–Hochberg correction. c RT-PCR analysis of NRF1 exon-7 inclusion in RNA from HAP1 cells transduced with three independent intergenic control hgRNAs or three independent hgRNAs targeting NRF1 exon-7 for deletion using scCHyMErA-Seq-compatible constructs. Representative images from three independent experiments. d qRT-PCR validation of NRF1 exon-7-dependent gene expression changes using the hgRNAs shown in Fig. 4c. Data represent mean ± SD from three biological replicates; **** p < 0.0001, *** p < 0.001, ** p < 0.01; two-way ANOVA with Dunnett’s multiple comparisons test. e Western blot analysis of NRF1 isoforms in HEK293 Flp-In cells stably expressing doxycycline-inducible 3×FLAG-tagged NRF1 with exon-7 included (FL) or excluded (ΔE7). The ectopic isoforms carry silent mutations at the siRNA target site, conferring resistance to siNRF1. Cells were transfected with control (siNT) or NRF1-targeting (siNRF1) siRNAs. Blots were probed for NRF1, FLAG, and GAPDH (loading control). Representative images from three independent experiments. f RNA-Seq analysis showing Z-score normalized expression changes of differentially expressed genes in siNRF1-treated cells rescued with NRF1-FL or -∆E7 isoforms. g ChIP-Seq density profiles of NRF1-FL and -∆E7 isoform enrichment over input at transcription start sites (TSS) ± 5 kb flanking regions of the genes regulated by NRF1 exon-7. h siRNA screen of 60 splicing regulators identifying factors controlling NRF1 exon-7 inclusion. Data represent the mean ± SD from three biological replicates; **** p < 0.0001, ** p < 0.01; two-way ANOVA.

Article Snippet: After fixation, cells were permeabilized in 0.2% Triton X-100 (Sigma-Aldrich #T8787) for 10 min, followed by blocking in 5% BSA (Sigma-Aldrich #A9647) for 1 h. Primary antibodies against NRF1 (1:500; Proteintech #12482-1-AP) and FLAG (1:500; Sigma-Aldrich #F3165) were applied for 1 h at room temperature in DPBS containing 5% BSA.

Techniques: Binding Assay, Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Transduction, Control, Construct, Quantitative RT-PCR, Biomarker Discovery, Gene Expression, Western Blot, Stable Transfection, Transfection, RNA Sequencing, ChIP-sequencing

Journal: Nature Communications

Article Title: Single-cell exon deletion profiling reveals splicing events that shape gene expression and cell state dynamics

doi: 10.1038/s41467-026-68774-w

Figure Lengend Snippet: a , c , e Venn diagrams showing the overlap of differentially expressed genes regulated by perturbation of NRF1 exon-7 ( a , c ) or TAF5 exon-8 ( e ). Overlaps are compared between our original scCHyMErA-Seq screen in HAP1 cells and either a small-scale replicate screen in HAP1 cells ( a ) or bulk RNA-Seq in HEK293 cells following isoform rescue experiments ( c , e ). Statistical significance was determined by two-sided Fisher’s exact test (**** p < 0.0001), and the corresponding odds ratios (OR) are indicated. b , d , f Scatterplots showing the Pearson correlation of log2 fold-changes for differentially expressed genes identified by scCHyMErA-Seq and either a replicate screen ( b ) or bulk RNA-Seq for NRF1 exon-7 ( d ) and TAF5 exon-8 ( f ) perturbations. Each point represents a gene; Two-sided Pearson correlation coefficients with corresponding significance values are indicated.

Article Snippet: After fixation, cells were permeabilized in 0.2% Triton X-100 (Sigma-Aldrich #T8787) for 10 min, followed by blocking in 5% BSA (Sigma-Aldrich #A9647) for 1 h. Primary antibodies against NRF1 (1:500; Proteintech #12482-1-AP) and FLAG (1:500; Sigma-Aldrich #F3165) were applied for 1 h at room temperature in DPBS containing 5% BSA.

Techniques: RNA Sequencing