Journal: GigaScience

Article Title: Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal

doi: 10.1093/gigascience/giaa045

Figure Lengend Snippet: Information regarding the possible resolution for various de novo genome sequencing technologies

Article Snippet: The 10x Genomics Chromium library was assembled using the 10x Genomics Supernova software [ ], using default parameters.

Techniques: Sequencing

Journal: GigaScience

Article Title: Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal

doi: 10.1093/gigascience/giaa045

Figure Lengend Snippet: Ten different assembly strategies using a variety of different data types: PCR-free Illumina short-read (“PCR-free”), long mate pair (“LMP”), 10x Genomics Chromium library (“10x”), and Bionano Genomics optical maps (“Bionano”). The blue-boxed assemblies all originate from the same PCR-free w2rap assembly (A1), and the black-boxed assemblies all originate from the same 10x Genomics Supernova assembly (A3). Information in paretheses refers to assembly software pipeline, and assembly numbers are annotated below each assembly.

Article Snippet: The 10x Genomics Chromium library was assembled using the 10x Genomics Supernova software [ ], using default parameters.

Techniques: Software

Journal: GigaScience

Article Title: Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal

doi: 10.1093/gigascience/giaa045

Figure Lengend Snippet: Genome assembly statistics (for sequences >1 kb) for all assemblies

Article Snippet: The 10x Genomics Chromium library was assembled using the 10x Genomics Supernova software [ ], using default parameters.

Techniques:

Journal: GigaScience

Article Title: Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal

doi: 10.1093/gigascience/giaa045

Figure Lengend Snippet: Comparison of the number of breaks introduced by REAPR for each of the technologies used to scaffold the w2rap-only assembly (A1)

Article Snippet: The 10x Genomics Chromium library was assembled using the 10x Genomics Supernova software [ ], using default parameters.

Techniques: Comparison

Journal: GigaScience

Article Title: Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal

doi: 10.1093/gigascience/giaa045

Figure Lengend Snippet: Comparison of the number of breaks introduced by REAPR for each of the technologies used to scaffold the 10x assembly (A3)

Article Snippet: The 10x Genomics Chromium library was assembled using the 10x Genomics Supernova software [ ], using default parameters.

Techniques: Comparison

Journal: GigaScience

Article Title: Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal

doi: 10.1093/gigascience/giaa045

Figure Lengend Snippet: Repeat content of assemblies

Article Snippet: The 10x Genomics Chromium library was assembled using the 10x Genomics Supernova software [ ], using default parameters.

Techniques:

Journal: GigaScience

Article Title: Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal

doi: 10.1093/gigascience/giaa045

Figure Lengend Snippet: Cumulative z -scores of assemblies (solid black circles). Error bars represent the minimum and maximum cumulative z -score after removing each metric in turn and recalculating the z -score for each assembly. Wide error bars show assemblies that are strongly affected by a given metric. For example, the 10x + lmp + bionano assembly (A8) has a long lower-boundary error bar because it has an exceptionally high scaffold N50 z -score (double that of the next nearest ranking assembly) and hence omitting this metric results in the assembly scoring much lower.

Article Snippet: The 10x Genomics Chromium library was assembled using the 10x Genomics Supernova software [ ], using default parameters.

Techniques:

Journal: GigaScience

Article Title: Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal

doi: 10.1093/gigascience/giaa045

Figure Lengend Snippet: REAPR statistics showing the percentage of error-free bases in the assembly, N50s before and after breaking at breakpoints, the percentage decrease in scaffold N50 after breaking, and the fragment coverage distribution (FCD) errors including errors across gaps

Article Snippet: The 10x Genomics Chromium library was assembled using the 10x Genomics Supernova software [ ], using default parameters.

Techniques:

Journal: Cells

Article Title: Identification and Characterization of ATOH7-Regulated Target Genes and Pathways in Human Neuroretinal Development

doi: 10.3390/cells13131142

Figure Lengend Snippet: Graphical summary of the study design. Human iPSCs were edited using a CRISPR/Cas9 approach. From the edited cell population, single cells were sorted and expanded in 96-well plates, followed by genotyping by Sanger sequencing. Selected clones were then differentiated to retinal organoids, which were then collected for different experiments. iPSC, induced pluripotent stem cell; WT, wildtype; KO, knockout; CUT&RUN-seq, cleavage under targets and release using nuclease sequencing; scRNA-seq, single-cell RNA sequencing; MUT-GFP, mutant reporter; WT-GFP, wildtype reporter.

Article Snippet: To gain better insights into the identities of retinal cells expressing ATOH7 and its target genes, we performed scRNA-seq on ATOH7 WT and ATOH7 KO week 7 whole-organoid dissociations using Chromium Fixed RNA Profiling (10X Genomics, Pleasanton, CA, USA).

Techniques: CRISPR, Sequencing, Clone Assay, Knock-Out, RNA Sequencing, Mutagenesis

Journal: Cells

Article Title: Identification and Characterization of ATOH7-Regulated Target Genes and Pathways in Human Neuroretinal Development

doi: 10.3390/cells13131142

Figure Lengend Snippet: Schematic representation of the iPSC cell lines used in the current study, which include isogenic WT and KO clones ( ATOH7 WT and ATOH7 KO ), as well as WT and mutant eGFP reporter lines ( ATOH7 WT-GFP and ATOH7 MUT-GFP ). iPSC, induced pluripotent stem cell; WT, wildtype; KO, knockout; UTR, untranslated region; CDS, coding sequence; T, c-Myc tag; eGFP, enhanced green fluorescent protein; P2A, porcine teschovirus-1 2A self-cleaving peptide; HA, homology arm; sgKO, single-guide RNA for ATOH7 knockout; PAM, protospacer-adjacent motif; MUT-GFP, mutant reporter; WT-GFP, wildtype reporter.

Article Snippet: To gain better insights into the identities of retinal cells expressing ATOH7 and its target genes, we performed scRNA-seq on ATOH7 WT and ATOH7 KO week 7 whole-organoid dissociations using Chromium Fixed RNA Profiling (10X Genomics, Pleasanton, CA, USA).

Techniques: Clone Assay, Mutagenesis, Knock-Out, Sequencing

Journal: Cells

Article Title: Identification and Characterization of ATOH7-Regulated Target Genes and Pathways in Human Neuroretinal Development

doi: 10.3390/cells13131142

Figure Lengend Snippet: Retinal organoid cell composition and cell type-specific genes regulated by ATOH7. Ten week 7 ATOH7 WT and ATOH7 KO retinal organoids were separately pooled, dissociated, and subsequently analyzed by hybridization-based PFA-fixed scRNA-seq. ( A ) UMAP of defined retinal clusters from integrated ATOH7 WT and ATOH7 KO organoids. The representative clusters are defined as naïve retinal progenitor cells ( nRPCs ), transient retinal progenitor cells ( tRPCs ), early retinal ganglion cells ( early RGCs ), late retinal ganglion cells ( late RGCs ), horizontal and amacrine cells ( H&As ), and photoreceptor cells ( PRs ). ( B ) Pseudotime trajectory analysis starting from TP53 -positive dividing retinal progenitor cells. ( C ) Separated UMAPs of cells derived from either ATOH7 WT or ATOH7 KO organoids. Percentages represent the proportion of cells in a given cluster compared to the total number of cells. Values in parentheses indicate the relative change in cell proportions compared to WT. ( D ) UMAP of re-clustered ATOH7 -positive cells derived from ATOH7 WT organoids. ( E ) ATOH7-targeted cell type marker genes, defined as scRNA-seq marker genes (AUC > 0.75) with a significant differential expression (adj. p -value < 0.01) between ATOH7 MUT-GFP and ATOH7 WT-GFP cells, based on reporter-enriched mRNA-seq, and at least one binding site discovered by CUT&RUN-seq, where the annotation is defined as either enhancer (>5 kb from TSS) or promoter (±5 kb from TSS). AUC, area under curve; CUT&RUN-seq, cleavage under targets and release using nuclease sequencing; KO, knockout; PFA, paraformaldehyde; scRNA-seq, single-cell RNA sequencing; TSS, transcription start site; UMAP, uniform manifold approximation and projection; DEGs, differentially expressed genes between MUT-GFP and WT-GFP; MUT-GFP, mutant reporter; WT-GFP, wildtype reporter; WT, wildtype.

Article Snippet: To gain better insights into the identities of retinal cells expressing ATOH7 and its target genes, we performed scRNA-seq on ATOH7 WT and ATOH7 KO week 7 whole-organoid dissociations using Chromium Fixed RNA Profiling (10X Genomics, Pleasanton, CA, USA).

Techniques: Hybridization, Derivative Assay, Marker, Quantitative Proteomics, Binding Assay, Sequencing, Knock-Out, RNA Sequencing, Mutagenesis

Journal: Cells

Article Title: Identification and Characterization of ATOH7-Regulated Target Genes and Pathways in Human Neuroretinal Development

doi: 10.3390/cells13131142

Figure Lengend Snippet: Axon guidance and Notch signaling are enriched amongst ATOH7 target DEGs. Identified ATOH7 target genes and ATOH7 -associated DEGs in eGFP-enriched reporter cells derived from week 7 retinal organoids were overlapped to identify differentially expressed ATOH7 target genes (ATOH7 target DEGs). ( A ) Venn diagram showing the identification of ATOH7 target DEGs. ( B ) Number of regulated genes per cell type amongst ATOH7 target DEGs, with cell-type specificity defined by scRNA-seq. The representative cell clusters are defined as naïve retinal progenitor cells ( nRPCs ), transient retinal progenitor cells ( tRPCs ), early retinal ganglion cells ( early RGCs ), late retinal ganglion cells ( late RGCs ), horizontal and amacrine cells ( H&As ), and photoreceptor cells ( PRs ). ( C ) Enrichment of GO terms amongst the 469 ATOH7 target DEGs compared to all annotated genes. The analysis was performed in g:Profiler and reduced by REVIGO, showing the top five most significant terms for biological processes ( GO:BP ), cellular compartments ( GO:CC ), and molecular function ( GO:MF ) for ATOH7 target DEGs. Additionally, the enrichment of biological pathway terms from combined KEGG, REACTOME, and WikiPathways databases was performed against all known human genes, showing top 15 terms according to adjusted p -value. Asterisks (*) demark significance (adj. p -value < 0.05) when including annotated genes only. Multiple correction testing was performed using g:SCS. ( D ) Selection of ATOH7 target DEGs intersecting with top significantly enriched biological pathways. Similar terms from different databases were grouped together. “Nervous System Development” (R-HSA-9675108) and “Axon Guidance” (R-HSA-422475) were grouped together due to an almost complete overlap (98%) of intersecting genes. Heatmaps of annotated genes present relative expression per cell type, based on scRNA-seq data of ATOH7 -positive cells in ATOH7 WT organoids, differential expression between ATOH7 MUT-GFP and ATOH7 WT-GFP cells, based on reporter-enriched mRNA-seq, and the number of annotated ATOH7-binding loci identified by CUT&RUN-seq, where the annotation is defined as either enhancer (>5 kb from TSS) or promoter (±5 kb from TSS). ( E ) Expression distribution of selected genes in cell type-specific scRNA-seq clusters. Violin plots show raw expression distribution for cells originating from ATOH7 WT organoids, ATOH7 KO organoids, and from the ATOH7 -expressing ( ATOH7 + ) cells, re-clustered from the ATOH7 WT organoids. Each expressing cell is marked by a black point. CUT&RUN-seq, cleavage under targets and release using nuclease sequencing; EVR, exudative vitreoretinopathy; g:SCS, g:Profiler set counts and sizes; GO, gene ontology; KO, knockout; REVIGO, reduce visualize gene ontology; PHPV, persistent hyperplastic primary vitreous; scRNA-seq, single-cell RNA sequencing; DEGs, differentially expressed genes between MUT-GFP and WT-GFP; MUT-GFP, mutant reporter; WT-GFP, wildtype reporter; TSS, transcription start site; WT, wildtype.

Article Snippet: To gain better insights into the identities of retinal cells expressing ATOH7 and its target genes, we performed scRNA-seq on ATOH7 WT and ATOH7 KO week 7 whole-organoid dissociations using Chromium Fixed RNA Profiling (10X Genomics, Pleasanton, CA, USA).

Techniques: Derivative Assay, Selection, Expressing, Quantitative Proteomics, Binding Assay, Sequencing, Knock-Out, RNA Sequencing, Mutagenesis

Journal: Cells

Article Title: Identification and Characterization of ATOH7-Regulated Target Genes and Pathways in Human Neuroretinal Development

doi: 10.3390/cells13131142

Figure Lengend Snippet: ATOH7 regulates genes encoding Wnt effectors and other secreted proteins. ( A ) Selection of ATOH7 reporter DEGs (log 2 FC > ±2, FDR < 0.01) encoding extracellular/secreted proteins. Heatmap of annotated genes present relative expression per cell type, based on scRNA-seq data, differential expression between ATOH7 MUT-GFP and ATOH7 WT-GFP cells, based on reporter-enriched mRNA-seq, and the number of annotated ATOH7-binding loci identified by CUT&RUN-seq, where the annotation is defined as either enhancer (>5 kb from TSS) or promoter (±5 kb from TSS). The representative cell clusters are defined as naïve retinal progenitor cells ( nRPCs ), transient retinal progenitor cells ( tRPCs ), early retinal ganglion cells ( early RGCs ), late retinal ganglion cells ( late RGCs ), horizontal and amacrine cells ( H&As ), and photoreceptor cells ( PRs ). ( B ) Expression distribution of selected genes encoding secreted proteins in cell type-specific scRNA-seq clusters. Violin plots show raw expression distribution in cells originating from ATOH7 WT organoids, ATOH7 KO organoids, and from the ATOH7 expressing ( ATOH7 + ) cells, re-clustered from the ATOH7 WT organoids. Each expressing cell is marked by a black dot. ( C ) Heatmap including the selection of ATOH7 reporter DEGs (log 2 FC > ±2, FDR < 0.01) associated with PHPV/EVR in patients ( LRP5 , NDP , KIF11 , and RCBTB1 ) and/or in vivo ( LRP5 , NDP , KIF11 , EFNA5 , EFNB2 , and NEO1 ) or in silico ( SMAD2 and, MYCN ) according to a literature search [ , , , , , , , , , , , , , , , , , , , , , , , , , , ]. ( D ) Expression distribution of genes encoding NDP and NDP -associated receptors in cell type-specific scRNA-seq clusters. CUT&RUN-seq , cleavage under targets and release using nuclease sequencing; EVR , exudative vitreoretinopathy; KO , knockout; PHPV , persistent hyperplastic primary vitreous; scRNA-seq , single-cell RNA sequencing; DEGs , differentially expressed genes between MUT-GFP and WT-GFP; MUT-GFP , mutant reporter; WT-GFP , wildtype reporter; TSS , transcription start site; WT , wildtype.

Article Snippet: To gain better insights into the identities of retinal cells expressing ATOH7 and its target genes, we performed scRNA-seq on ATOH7 WT and ATOH7 KO week 7 whole-organoid dissociations using Chromium Fixed RNA Profiling (10X Genomics, Pleasanton, CA, USA).

Techniques: Selection, Expressing, Quantitative Proteomics, Binding Assay, In Vivo, In Silico, Sequencing, Knock-Out, RNA Sequencing, Mutagenesis