ngn2 coding sequence  (Addgene inc)

Journal: Nature Cell Biology

Article Title: An activity-specificity trade-off encoded in human transcription factors

doi: 10.1038/s41556-024-01411-0

Figure Lengend Snippet: a . (left) Schematic models of NGN2 proteins. (middle) Omega plots and Ω Aro scores of the IDRs. (right) Results of luciferase reporter assays. Luciferase values were normalized against an internal Renilla control, and the values are displayed as percentages normalized to the activity measured using an empty vector (dashed orange line). Data are displayed as mean ± SD from three biological replicates. b . Representative images of droplet formation of purified NGN2 C-terminal IDR–mEGFP proteins. Scale bar: 5 μm. c . The relative amount of condensed protein per concentration quantified in the droplet formation assays. Data are displayed as mean ± SD. N = 10 images per condition pooled from two independent replicates. The curve was generated as a nonlinear regression to a sigmoidal curve function. d . Fluorescence microscopy images of differentiating ZIP13K2 cells expressing FLAG-tagged versions of NGN2 at 48 h. NGN2-FLAG was visualized with an α-FLAG antibody. GFP signal is the endogenous mEGFP fluorescence signal of mEGFP. Scale bar: 5 μm. e . Quantification of FLAG-NGN2 signal. Data displayed as mean ± SD. N = number of cells from one biological replicate. P values are from two-sided unpaired t-test. P (Wild type vs. AroLITE) =0.00001, P (Wild type vs. AroPERFECT) =0.00019. f . Heatmap analysis of RNA-Seq data in the four cell lines. Genes were clustered using k-means clustering on expression values. Expression values are represented by scaling and centering VST transformed read count normalized values (z-score). g . Marker gene analysis from selected genes from single-cell cluster markers in NGN2 induced neural differentiation. h . Principal component analysis of the NGN2 ChIP–Seq peak profiles. i . NGN2 AroLITE loss of binding at the SERTM1 locus. Displayed are genome browser tracks of ChIP–Seq data of NGN2 wild type, AroLITE and AroPERFECT in ZIP13K2 cells, 24 and 48 hours after NGN2 overexpression. Coordinates are hg38 genome assembly coordinates. j . Enrichment scores of bHLH TF motifs, and adjusted P values. P values from Benjamini–Hochberg method. k . Heatmap analysis of TT-SLAM-seq data in the four cell lines 12 h and 24 h after transgene induction. Genes were clustered using k-means clustering on expression values. Expression values are represented by scaling and centering VST transformed read count normalized values (z-score). l . TT-SLAM-Seq data at the LBH l ocus.

Article Snippet: N-terminally FLAG-tagged coding sequences of human wild-type, AroLITE or AroPERFECT NGN2 (Twist Bioscience) with a downstream T2A tag (Sigma) were cloned into a backbone of the inducible Caspex expression vector linearized by restriction digest with NcoI (NEB) and KpnI (NEB).

Techniques: Luciferase, Control, Activity Assay, Plasmid Preparation, Purification, Concentration Assay, Generated, Fluorescence, Microscopy, Expressing, RNA Sequencing Assay, Transformation Assay, Marker, ChIP-sequencing, Binding Assay, Over Expression

Journal: Nature Cell Biology

Article Title: An activity-specificity trade-off encoded in human transcription factors

doi: 10.1038/s41556-024-01411-0

Figure Lengend Snippet: a . (left) Western blot of GAL4-DBD and GAL4-DBD-MYOD1 C-IDR-fusion proteins in HEK293T cells 24 hours after transfection using a GAL4-DBD specific antibody. (left). Western blot of FLAG-MYOD1 fusion proteins in differentiating C2C12 cells 24 hours after transgene induction. Wild type and AroPERFECT mutants are expressed at comparable levels. HSP90: loading control. Wild type and AroPERFECT mutants are expressed at comparable levels. b . Results of a MYOD1 C-IDR tiling experiment by using luciferase reporter assays. Sequences were tiled into fragments of 40 amino acids with 20 amino acid overlaps. Data displayed as mean ± SD. N = 2 biological replicates. The activities of the full-length IDRs are indicated with dashed horizontal lines. c . Fluorescence images of C2C12 myoblasts at day 0 and 1 after induction of MYOD1 wild type, MYOD1 AroLITE, MYOD1 AroPERFECT C or MYOD1 AroLITE C transgene with doxycycline. DAPI was used as DNA counterstain (magenta). Co-expressed mEGFP of the MYOD1-T2A-mEGFP fusion protein was used as cytoplasmic marker (cyan). Scale bar 0.5 mm. d . Principal component analysis of the RNA-Seq expression profiles of Parental C2C12, C2C12 MYOD1 wild type, C2C12 MYOD1 AroLITE, C2C12 MYOD1 AroPERFECT, C2C12 MYOD1 AroLITE C and C2C12 MYOD1AroPERFECT-C cells (PC1 vs. PC2). e . Differential expression analysis of Parental C2C12 (top), C2C12 MYOD1 AroPERFECT (centre) and C2C12 MYOD1 AroLITE C (bottom) cells versus C2C12 MYOD1 wild type cells. MYOD1 target genes are highlighted in blue. P -values from Benjamini–Hochberg method. f . Heatmap analysis of RNA-Seq data in the six cell lines. Genes were clustered using k-means clustering on expression values. Expression values are represented by scaling and centering VST transformed read count normalized values (z-score). K-means clustering was used to define the clusters. g . Gene set enrichment analysis (GSEA) of differentially expressed genes in the MYOD1 AroPERFECT C RNA-Seq sample. Empirical P value is reported.

Article Snippet: N-terminally FLAG-tagged coding sequences of human wild-type, AroLITE or AroPERFECT NGN2 (Twist Bioscience) with a downstream T2A tag (Sigma) were cloned into a backbone of the inducible Caspex expression vector linearized by restriction digest with NcoI (NEB) and KpnI (NEB).

Techniques: Western Blot, Transfection, Control, Luciferase, Fluorescence, Marker, RNA Sequencing Assay, Expressing, Transformation Assay

Journal: Nature Cell Biology

Article Title: An activity-specificity trade-off encoded in human transcription factors

doi: 10.1038/s41556-024-01411-0

Figure Lengend Snippet: a . Representative images of fluorescence recovery after photobleaching (FRAP) experiments with HOXD4 IDR–mEGFP droplets. b . Western blot of GAL4-DBD and GAL4-DBD-HOXD4-IDR-fusion proteins in HEK293T cells 24 hours after transfection using a GAL4-DBD specific antibody. HSP90: loading control. Except for AroLITE A, GAL4-DBD-HOXD4-IDR fusion proteins are expressed at comparable levels. c . Schematic models of HOXD4 wild type and mutant IDRs. Omega plots of the HOXD4 IDRs and Ω Aro scores are shown next to the schematic models. d . Results of luciferase reporter assays. The YPWM motif does not contribute to the transactivation potential of the HOXD4 IDR. e . The activity of HOXD4 IDRs (left) and C/EBPα IDRs (right) scales with the number of small inert residues adjacent to aromatic residues in the IDR constructs. f . (left) Schematic models of wild type and AroPERFECT HOXC4 IDRs. (middle) Omega plots and Ω Aro scores of the IDRs. IDR: intrinsically disordered region (right). Results of luciferase reporter assays. g . Western blot of GAL4-DBD and GAL4-DBD-HOXC4-IDR fusion proteins in HEK293T cells 24 hours after transfection using a GAL4-DBD specific antibody. HSP90: loading control. h . Representative images of droplet formation of purified HOXC4 IDR–mEGFP proteins. Scale bar: 5 μm. For the wild type IDR, the exact same images are displayed in Fig. . i . The relative amount of condensed protein per concentration quantified in the droplet formation assays. Data are displayed as mean ± SD. N = 10 images per condition pooled from two independent replicates. The curve was generated as a nonlinear regression to a sigmoidal curve function. j . Representative images FRAP experiments with HOXC4 IDR–mEGFP droplets. k . Fluorescence intensity of HOXC4 wild type IDR and HOXC4 AroPERFECT IDR in vitro droplets before, during and after photobleaching. Data displayed as mean ± SD. N = 20 images from two replicates. In d ., f . luciferase values were normalized against an internal Renilla control, and the values are displayed as percentages normalized to the activity measured using an empty vector. Data are displayed as mean ± SD from three biological replicates. P values are from two-sided unpaired t-tests.

Article Snippet: N-terminally FLAG-tagged coding sequences of human wild-type, AroLITE or AroPERFECT NGN2 (Twist Bioscience) with a downstream T2A tag (Sigma) were cloned into a backbone of the inducible Caspex expression vector linearized by restriction digest with NcoI (NEB) and KpnI (NEB).

Techniques: Fluorescence, Western Blot, Transfection, Control, Mutagenesis, Luciferase, Activity Assay, Construct, Purification, Concentration Assay, Generated, In Vitro, Plasmid Preparation

Journal: Nature Cell Biology

Article Title: An activity-specificity trade-off encoded in human transcription factors

doi: 10.1038/s41556-024-01411-0

Figure Lengend Snippet: a , Schematic models of wild-type and mutant C/EBPα proteins (left). The positions of the bZIP DBD (grey box) and aromatic residues (orange dots) are indicated. Omega plots and Ω Aro scores (middle). Results of luciferase reporter assays (right). Data are the mean ± s.d. of n = 3 biological replicates with three technical replicates each. b , Representative images of droplet formation of purified C/EBPα IDR–mEGFP fusion proteins at the indicated concentrations in droplet formation buffer. Scale bars, 5 μm. c , Fluorescence intensity of C/EBPα wild type, AroLITE and AroPERFECT IS15 IDR in in vitro droplets before, during and after photobleaching. Data are the mean ± s.d. of n = 15 (wild-type) and 14 (AroPERFECT IS15 and AroPERFECT IS10) droplets from two replicates. d , Fluorescence images of ectopically expressed YFP–RNAPII CTD in live U2OS cells that were cotransfected with the indicated CFP–LacI-C/EBPα IDR fusion constructs. The dashed line represents the nuclear contour. Inserts: magnified views of the regions in the red boxes. Scale bars, 10 μm (main images) and 40 μm (inserts). e , Relative YFP signal intensity in the tether foci. Data are the mean ± s.d. of n = 51 (wild-type YFP, AroPERFECT YFP and wild-type YFP–RNAPII CTD) and 56 (AroPERFECT YFP–RNAPII CTD) nuclei pooled from two independent replicates. f , Results of a C/EBPα IDR tiling experiment using luciferase reporter assays. C/EBPα wild type and AroPERFECT IS15 IDR sequences were tiled into fragments of 40 amino acids with 20-amino-acid overlaps. The activities of the full-length IDRs are indicated with dashed horizontal lines. g , Results of luciferase reporter assays of the indicated IDR constructs. a , f , g , Luciferase values were normalized to an internal Renilla control and the values are displayed as percentages normalized to the activity measured using an empty vector. f , g , Data are the mean ± s.d. of n = 3 biological replicates. a , e , g , P values are from a two-sided unpaired Student’s t -tests.

Article Snippet: N-terminally FLAG-tagged coding sequences of human wild-type, AroLITE or AroPERFECT NGN2 (Twist Bioscience) with a downstream T2A tag (Sigma) were cloned into a backbone of the inducible Caspex expression vector linearized by restriction digest with NcoI (NEB) and KpnI (NEB).

Techniques: Mutagenesis, Luciferase, Purification, Fluorescence, In Vitro, Construct, Control, Activity Assay, Plasmid Preparation

Journal: Nature Cell Biology

Article Title: An activity-specificity trade-off encoded in human transcription factors

doi: 10.1038/s41556-024-01411-0

Figure Lengend Snippet: a , Schematic models of wild-type and mutant C/EBPα proteins. The transactivation data are identical to the data displayed in Fig. . P values are from two-sided unpaired Student’s t -tests. b , Schematic model of C/EBPα-mediated transdifferentiation of B cells to macrophages. c , FACS quantification of GFP + RCH-rtTA cells encoding C/EBPα overexpression cassettes. The proportions of CD19 − Mac1 + cells were measured 48, 96 and 168 h after transgene induction. Data are the mean ± s.d. of n = 5 (wild type and AroPERFECT IS15) and 3 (AroLITE and AroPERFECT IS10) independent experiments. d , Graph-based clustering (uniform manifold approximation and projection, UMAP) of the scRNA-seq data of C/EBPα-mediated transdifferentiation. Clusters were annotated based on marker genes. Overlayed is the partition-based graph abstraction (PAGA) showing the cell trajectory based on dynamic modelling of RNA velocity. Inset: pseudotime plot. e , Proportion of mEGFP + cells in the macrophage clusters (colour-coded as in d ). f , Heatmap representation of ChIP–Seq read densities of wild-type and AroPERFECT IS15 C/EBPα within a 1.5-kb window around all shared C/EBPα peaks and differentially enriched peaks in AroPERFECT IS15 C/EBPα. ‘Peaks unique to IS15 and reported before’ denotes binding sites differentially enriched in IS15 binding that overlap C/EBPα peaks reported in previous literature. FE, fold enrichment. g , Enrichment scores of bZIP TF motifs and adjusted (adj.) P values of enrichment at the three indicated peak sets. P values were determined using the Benjamini–Hochberg method. h , j , AroPERFECT IS15 C/EBPα shows enhanced binding at the FAM98A ( h ) and GBP5 ( j ) loci. Displayed are genome browser tracks of ChIP–Seq data of C/EBPα 24 and 48 h after C/EBPα induction. The coordinates are hg38 genome assembly coordinates. i , k , UMAPs coloured on FAM98A ( i ) and GBP5 ( k ) expression. The numbers denote the mean ± s.d. expression in the whole samples. l , Luciferase assays using the indicated reporter plasmids cotransfected with expression vectors encoding either wild-type or AroPERFECT IS15 C/EBPα. Luciferase values were normalized to an internal Renilla control and the values are displayed as percentages of the activity measured using the ‘basic’ vector. Data are the mean ± s.d. of four biological replicates. P values are from two-sided unpaired Student’s t -tests.

Article Snippet: N-terminally FLAG-tagged coding sequences of human wild-type, AroLITE or AroPERFECT NGN2 (Twist Bioscience) with a downstream T2A tag (Sigma) were cloned into a backbone of the inducible Caspex expression vector linearized by restriction digest with NcoI (NEB) and KpnI (NEB).

Techniques: Mutagenesis, Over Expression, Marker, ChIP-sequencing, Binding Assay, Expressing, Luciferase, Control, Activity Assay, Plasmid Preparation

Journal: Nature Cell Biology

Article Title: An activity-specificity trade-off encoded in human transcription factors

doi: 10.1038/s41556-024-01411-0

Figure Lengend Snippet: a , Schematic models of wild-type and mutant NGN2 proteins (left). The positions of the bHLH DBD (grey box) and aromatic amino acids (yellow dots) are indicated. Omega plots and Ω Aro scores (right). b , Fluorescence intensity of NGN2 wild-type and AroPERFECT IDR in in vitro droplets before, during and after photobleaching. Data are the mean ± s.d. of n = 20 droplets pooled from two independent replicates. c , Schematic model of the NGN2-mediated human iPSC-to-neuron differentiation experiment. ROCKi, Rho-kinase inhibitor. d , Representative fluorescence microscopy images of differentiating human iPSCs expressing the indicated NGN2 proteins. Hoechst dye was used as a nuclear counterstain; mEGFP, NGN2-T2A–mEGFP. Insets: magnified views of the regions in the white boxes. Scale bars, 0.1 mm (main images) and 0.05 mm (insets). e , Number of cells, based on Hoechst nuclear staining, in the NGN2-directed differentiation experiments. f , Neurite density (fraction of tubulin-covered area) in the NGN2-directed differentiation experiments. e , f , Data are the mean ± s.d. of n = 6 images pooled from two independent experiments. P values from a two-sided unpaired Student’s t -test. g , Principal component analysis of the RNA-seq expression profiles of parental ZIP13K2 human iPSCs and human iPSCs expressing the indicated NGN2 transgenes. h , Differential expression analysis of human iPSCs expressing the indicated transgenes. NGN2 target genes are highlighted. P values were determined using the Benjamini–Hochberg method. i , Heatmap representation of ChIP–Seq read densities of cells expressing wild-type, AroLITE and AroPERFECT NGN2 within a 1.5 kb window around all shared NGN2 peaks (top), differentially enriched peaks in AroPERFECT NGN2 (centre) and differentially enriched peaks in wild-type NGN2 (bottom). FE, fold over input. j , NGN2 differential binding at the TMEM97 locus. Genome browser tracks of ChIP–Seq data after 24 and 48 h of NGN2 expression are displayed. The arrowhead highlights a differentially bound peak at 24 h. The coordinates are hg38 genome assembly coordinates. k , Nascent transcription (TT-SLAM-Seq) metagene profiles at approximately 9,000 NGN2 target genes. TSS, transcription start site; TES, transcription end site.

Article Snippet: N-terminally FLAG-tagged coding sequences of human wild-type, AroLITE or AroPERFECT NGN2 (Twist Bioscience) with a downstream T2A tag (Sigma) were cloned into a backbone of the inducible Caspex expression vector linearized by restriction digest with NcoI (NEB) and KpnI (NEB).

Techniques: Mutagenesis, Fluorescence, In Vitro, Microscopy, Expressing, Staining, RNA Sequencing, Quantitative Proteomics, ChIP-sequencing, Binding Assay

Journal: Nature Cell Biology

Article Title: An activity-specificity trade-off encoded in human transcription factors

doi: 10.1038/s41556-024-01411-0

Figure Lengend Snippet: a . (left) Schematic models of NGN2 proteins. (middle) Omega plots and Ω Aro scores of the IDRs. (right) Results of luciferase reporter assays. Luciferase values were normalized against an internal Renilla control, and the values are displayed as percentages normalized to the activity measured using an empty vector (dashed orange line). Data are displayed as mean ± SD from three biological replicates. b . Representative images of droplet formation of purified NGN2 C-terminal IDR–mEGFP proteins. Scale bar: 5 μm. c . The relative amount of condensed protein per concentration quantified in the droplet formation assays. Data are displayed as mean ± SD. N = 10 images per condition pooled from two independent replicates. The curve was generated as a nonlinear regression to a sigmoidal curve function. d . Fluorescence microscopy images of differentiating ZIP13K2 cells expressing FLAG-tagged versions of NGN2 at 48 h. NGN2-FLAG was visualized with an α-FLAG antibody. GFP signal is the endogenous mEGFP fluorescence signal of mEGFP. Scale bar: 5 μm. e . Quantification of FLAG-NGN2 signal. Data displayed as mean ± SD. N = number of cells from one biological replicate. P values are from two-sided unpaired t-test. P (Wild type vs. AroLITE) =0.00001, P (Wild type vs. AroPERFECT) =0.00019. f . Heatmap analysis of RNA-Seq data in the four cell lines. Genes were clustered using k-means clustering on expression values. Expression values are represented by scaling and centering VST transformed read count normalized values (z-score). g . Marker gene analysis from selected genes from single-cell cluster markers in NGN2 induced neural differentiation. h . Principal component analysis of the NGN2 ChIP–Seq peak profiles. i . NGN2 AroLITE loss of binding at the SERTM1 locus. Displayed are genome browser tracks of ChIP–Seq data of NGN2 wild type, AroLITE and AroPERFECT in ZIP13K2 cells, 24 and 48 hours after NGN2 overexpression. Coordinates are hg38 genome assembly coordinates. j . Enrichment scores of bHLH TF motifs, and adjusted P values. P values from Benjamini–Hochberg method. k . Heatmap analysis of TT-SLAM-seq data in the four cell lines 12 h and 24 h after transgene induction. Genes were clustered using k-means clustering on expression values. Expression values are represented by scaling and centering VST transformed read count normalized values (z-score). l . TT-SLAM-Seq data at the LBH l ocus.

Article Snippet: N-terminally FLAG-tagged coding sequences of human wild-type, AroLITE or AroPERFECT NGN2 (Twist Bioscience) with a downstream T2A tag (Sigma) were cloned into a backbone of the inducible Caspex expression vector linearized by restriction digest with NcoI (NEB) and KpnI (NEB).

Techniques: Luciferase, Control, Activity Assay, Plasmid Preparation, Purification, Concentration Assay, Generated, Fluorescence, Microscopy, Expressing, RNA Sequencing, Transformation Assay, Marker, ChIP-sequencing, Binding Assay, Over Expression

Journal: Nature Cell Biology

Article Title: An activity-specificity trade-off encoded in human transcription factors

doi: 10.1038/s41556-024-01411-0

Figure Lengend Snippet: a , Schematic models of wild-type and mutant MYOD1 proteins (left). The position of the bHLH DBD (grey box) and aromatic amino acids (orange dots) are indicated. Omega plots and Ω Aro scores of the N-terminal and C-terminal IDRs (middle). Results of luciferase reporter assays in C2C12 mouse myoblasts (right). Luciferase values were normalized to an internal Renilla control and the values are displayed as percentages normalized to the activity measured using an empty vector. Data are the mean ± s.d. of three biological replicates. P values are from two-sided unpaired Student’s t -tests. b , Schematic model of the MYOD1-mediated myotube differentiation experiment. c , Representative fluorescence microscopy images of differentiating C2C12 myoblasts expressing the indicated MYOD1 proteins on day 3 after DOX induction. The mEGFP signal of the MYOD1-T2A–mEGFP construct was used as a cytoplasmic marker. Nuclear counterstain (DAPI) is shown in magenta. Magnified views of the regions in the white boxes are provided (zoom; bottom). Scale bars, 0.5 mm (main images) and 0.2 mm (zoom). d , MYOD1-driven myotube differentiation efficiency. The fusion index was calculated as the percentage of nuclei in fused cells (cells containing at least three nuclei). Data are the mean ± s.d. of n = 15 images per genotype pooled from three biological replicates. P values are from two-sided unpaired Student’s t -tests. e , Principal component analysis of RNA-seq expression profiles of parental C2C12 cells as well as cells expressing the indicated MYOD1 transgenes. f , Differential expression analysis of C2C12 cells expressing AroLITE or AroPERFECT C MYOD1 versus C2C12 cells expressing wild-type MYOD1. MYOD1 target genes are represented as blue dots. Highlighted genes were differentially expressed and are involved in cell adhesion. P values were calculated using the Benjamini–Hochberg method.

Article Snippet: N-terminally FLAG-tagged coding sequences of human wild-type, AroLITE or AroPERFECT NGN2 (Twist Bioscience) with a downstream T2A tag (Sigma) were cloned into a backbone of the inducible Caspex expression vector linearized by restriction digest with NcoI (NEB) and KpnI (NEB).

Techniques: Mutagenesis, Luciferase, Control, Activity Assay, Plasmid Preparation, Fluorescence, Microscopy, Expressing, Construct, Marker, RNA Sequencing, Quantitative Proteomics

Journal: Nature Cell Biology

Article Title: An activity-specificity trade-off encoded in human transcription factors

doi: 10.1038/s41556-024-01411-0

Figure Lengend Snippet: a . (left) Western blot of GAL4-DBD and GAL4-DBD-MYOD1 C-IDR-fusion proteins in HEK293T cells 24 hours after transfection using a GAL4-DBD specific antibody. (left). Western blot of FLAG-MYOD1 fusion proteins in differentiating C2C12 cells 24 hours after transgene induction. Wild type and AroPERFECT mutants are expressed at comparable levels. HSP90: loading control. Wild type and AroPERFECT mutants are expressed at comparable levels. b . Results of a MYOD1 C-IDR tiling experiment by using luciferase reporter assays. Sequences were tiled into fragments of 40 amino acids with 20 amino acid overlaps. Data displayed as mean ± SD. N = 2 biological replicates. The activities of the full-length IDRs are indicated with dashed horizontal lines. c . Fluorescence images of C2C12 myoblasts at day 0 and 1 after induction of MYOD1 wild type, MYOD1 AroLITE, MYOD1 AroPERFECT C or MYOD1 AroLITE C transgene with doxycycline. DAPI was used as DNA counterstain (magenta). Co-expressed mEGFP of the MYOD1-T2A-mEGFP fusion protein was used as cytoplasmic marker (cyan). Scale bar 0.5 mm. d . Principal component analysis of the RNA-Seq expression profiles of Parental C2C12, C2C12 MYOD1 wild type, C2C12 MYOD1 AroLITE, C2C12 MYOD1 AroPERFECT, C2C12 MYOD1 AroLITE C and C2C12 MYOD1AroPERFECT-C cells (PC1 vs. PC2). e . Differential expression analysis of Parental C2C12 (top), C2C12 MYOD1 AroPERFECT (centre) and C2C12 MYOD1 AroLITE C (bottom) cells versus C2C12 MYOD1 wild type cells. MYOD1 target genes are highlighted in blue. P -values from Benjamini–Hochberg method. f . Heatmap analysis of RNA-Seq data in the six cell lines. Genes were clustered using k-means clustering on expression values. Expression values are represented by scaling and centering VST transformed read count normalized values (z-score). K-means clustering was used to define the clusters. g . Gene set enrichment analysis (GSEA) of differentially expressed genes in the MYOD1 AroPERFECT C RNA-Seq sample. Empirical P value is reported.

Article Snippet: N-terminally FLAG-tagged coding sequences of human wild-type, AroLITE or AroPERFECT NGN2 (Twist Bioscience) with a downstream T2A tag (Sigma) were cloned into a backbone of the inducible Caspex expression vector linearized by restriction digest with NcoI (NEB) and KpnI (NEB).

Techniques: Western Blot, Transfection, Control, Luciferase, Fluorescence, Marker, RNA Sequencing, Expressing, Quantitative Proteomics, Transformation Assay