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Sample GSM7877350 Query DataSets for GSM7877350
Status Public on Jan 01, 2024
Title Mutant, E11.5, Sorted CNCCs
Sample type SRA
 
Source name Wnt1-CRE recombined cells (i.e., eGFP+) cell sorted from dissocated E11.5 heads (rostral to the otic placode)
Organism Mus musculus
Characteristics tissue: Wnt1-CRE recombined cells (i.e., eGFP+) cell sorted from dissocated E11.5 heads (rostral to the otic placode)
genotype: Tfap2a/Tfap2b neural crest mutant (Tfap2a flox/flox; Tfap2b flox/flox; r26 mTmG/mTmG; Wnt1CRE+)
Extracted molecule polyA RNA
Extraction protocol Single-cell dissociation, cell sorting, and sequencing: To isolate CNCCs, E11.5 embryonic heads were cut just below pharyngeal arch 2 and the otic vesicle by microdissection. Using Wnt1:CRE-mediated recombination of the mTmG reporter allele (Muzumdar et al., 2007), CRE-positive embryos were selected for further processing. Tissue samples were then subject to a cold protease single-cell dissociation protocol (Adam et al., 2017), with modifications. Embryo heads were incubated in a protease cocktail comprised of Bacillus licheniformis Subtilisin A (Creative Enzymes), Accumax, and Accutase (Fisher Scientific) suspended in DEPC-PBS as previously described (Sekiguchi and Hauser, 2019). The tissue was then carefully disrupted using a disposable Eppendorf pellet pestle and placed on a rotator at 4°C for 30 min. During this step, samples were gently triturated every 10 min using a wide bore 1 mL pipette tip. The enzymatic reaction was quenched using an equal volume of 10% heat-inactivated FBS (Fisher Scientific) in 1X phenol red-free DMEM (ThermoFisher). Dissociated cells were washed thrice in DEPC-PBS by centrifugation at 600 x g at 4°C for 5 min and then transferred, through Bel Art SP Scienceware Flowmi 40-μm strainers (ThermoFisher), into chilled flow tubes precoated with DEPC-PBS. After GFP-positive sorting on the University of Iowa Flow Cytometry Facility’s ARIA II system (Becton Dickinson), CNCCs were re-suspended in DEPC-PBS containing 0.04% non-acetylated BSA (New England Biolabs). Cell viability was determined to be >95% by Trypan Blue staining. During cell sorting, genotypes were confirmed by rapid genotyping, allowing isolation of GFP-positive cells from two Tfap2HET control and two sibling-matched Tfap2NCKO mutant embryos. Cells from each “biological replicate” were combined to generate a single control and mutant sample. ~6,000 GFP-positive CNCCs from each condition were subject to library preparation by the University of Iowa Genomics Division on the 3’ expression scRNA-seq 10X Chromium v3.1 pipeline. Libraries were sequenced on an Illumina NovaSeq 6000 platform as 100-bp paired-end reads. Over 20,000 reads per cell were acquired, resulting in ~1.5 x 108 reads per condition.
 
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description 10x Genomics
Data processing Quality control, integration, and UMAP visualization: Sequencing results were demultiplexed and converted to a FASTQ format using the Illumina bcl2fastq software. Reads were processed and aligned to the mm10 reference genome assembly with the Cell Ranger Count function. Seurat v4.3.0 was used for quality control, filtering out cells with >10% mitochondrial counts, less than 200 features, and more than 7,500 features. We then integrated Tfap2HET and Tfap2NCKO conditions (Hao et al., 2021), where RNA counts were normalized and FindVariableFeatures was run with the following parameters: selection.method = “vst”, nfeatures = 2,000. Cell clustering by Uniform Manifold Approximation and Projection (UMAP) plots was deployed by RunPCA and then RunUMAP (dims 1:30). The UMAP visualizing all CNCCs was generated with FindClusters using a resolution of 0.40. Major CNCC derivatives were annotated based on published immunofluorescence and scRNA-seq profiling (Soldatov et al., 2019). Gene expression was plotted on UMAP plots by FeaturePlot. Gene expression mapping of CNCC lineage markers was done using the FeaturePlot function. Gene expression-based cell cycle analysis: The CellCycleScoring function was performed as previously described, with minor modifications (Hao et al., 2021). The UMAP resolution was changed to 0.05 to segregate the major CNCC lineages as individual clusters (i.e., mesenchyme, neuron, glia). Bioconductor BiomaRt v2.46.3 (Durinck et al., 2005) was used to convert a published list of cell cycle genes (Kowalczyk et al., 2015) from human to mouse gene nomenclature. Cell cycle scores were then mapped directly onto the UMAP, while GraphPad Prism was used to graph the scores as a percentage relative to each cluster. Cellular distribution analysis: To examine genotype cellular distributions in each cluster, each condition was expressed as a percentage per cluster. These analyses were visualized in GraphPad Prism. For the initial UMAP containing all sorted cells, a resolution of 0.05 was used. For the MAGIC-clustered UMAP, a resolution of 0.10 was used (see “Annotation of mesenchyme positional identities”). Annotation of mesenchyme positional identities: To better define the facial prominence subpopulations, we subset and re-clustered the “Mesenchyme” clusters using MAGIC v2.0.3 (van Dijk et al., 2018) with RunUMAP dims 1:20. Major mesenchyme populations were then annotated based on known gene markers, which were plotted on the MAGIC-clustered UMAP with DefaultAssay = “MAGIC_RNA”. A cluster resolution of 0.10 was chosen to best visualize each prominence population and the second pharyngeal arch as individual clusters based on published in situ data, ChIP-seq, ATAC-seq, and transcriptomic datasets (Gu et al., 2022; Hooper et al., 2020; Minoux et al., 2017). To visualize co-expression of two genes on the UMAP, we used FeaturePlot (flag blend = “TRUE”). Gene-set and gene expression analyses: The UMAP resolution was set to 0.05 to distinguish the major cell groupings, to which we then subset the “pseudobulked” mesenchyme group. We then used the FindMarkers function with ident.1 = Tfap2HET and ident.2 = Tfap2NCKO on CNCCs, to generate a list of differentially expressed genes (adjusted p-value < 0.05 and a log fold-change threshold of 0.10) (Table S2, Tab 9). For gene-set enrichment, ontology, and pathway analysis, lists of either up- or downregulated genes, generated from the mesenchyme “pseudobulk” approach, in the Tfap2NCKO condition were inputted in the Enrichr pipeline. Control-versus-mutant expression analysis of select genes was visualized as a violin plot using the VlnPlot function on the MAGIC-clustered UMAP, with DefaultAssay = “RNA”. Differential gene expression was validated for a select set of genes-of-interest by targeted approaches (see real-time PCR).
Assembly: mm10
 
Submission date Nov 01, 2023
Last update date Jan 01, 2024
Contact name Eric Van Otterloo
E-mail(s) eric-vanotterloo@uiowa.edu
Organization name University of Iowa
Street address 801 Newton Rd
City Iowa City
State/province Iowa
ZIP/Postal code 52242
Country USA
 
Platform ID GPL24247
Series (2)
GSE246806 TFAP2 paralogs regulate midfacial development in part through a conserved ALX genetic pathway [scRNA-seq]
GSE246807 TFAP2 paralogs regulate midfacial development in part through a conserved ALX genetic pathway
Relations
BioSample SAMN38066129
SRA SRX22332093

Supplementary file Size Download File type/resource
GSM7877350_AB_head_filtered_barcodes.tsv.gz 31.0 Kb (ftp)(http) TSV
GSM7877350_AB_head_filtered_features.tsv.gz 284.1 Kb (ftp)(http) TSV
GSM7877350_AB_head_filtered_matrix.mtx.gz 79.2 Mb (ftp)(http) MTX
SRA Run SelectorHelp
Raw data are available in SRA

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