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| Status |
Public on Jun 12, 2024 |
| Title |
C3H dual-lineage receiver, Day 3, No ligand, rep 2 |
| Sample type |
SRA |
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| Source name |
c3h/10t1/2
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| Organism |
Mus musculus |
| Characteristics |
cell line: c3h/10t1/2
genotype: SFFV_myc- LaG17_synNotch_TetRVP64 , TRE_MyoD-P2A-miRFP, EF1a_flag-LaM4_synNotch_Gal4-VP64 , UAS_ETV2-P2A-tBFP_PGK_HygromycinR
treatment: No ligand
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| Treatment protocol |
All cells were droplet seeded onto Ibidi wells patterned using capillary fluidic device with No ligand, GFP rows, mcherry rows, or both GFP and mCherry interdigiting rows and cultured for 3 days before single nuclei RNA extraction and sequencing. Droplet seeding was done as follows: 3.3x10^4 dual-lineage cells were seeded in a 30uL droplet on top of the patterns, allowed to attach for 30 minutes, prior to adding additional media.
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| Growth protocol |
Cells were maintained in high glucose DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C
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| Extracted molecule |
total RNA |
| Extraction protocol |
cells were trypsinized and cells were lysed in IGEPAL CA630-containing lysis buffer for 7 minutes to isolate individual nuclei.
Library construction was performed according to the manufacturer’s protocol (10x Genomics single cell 3’ v3.1 protocol). Briefly, after resuspension and counting, 16,000 GCs per experiment were resuspended in master mix and loaded (together with partitioning oil and gel beads), onto each lane of an 8 lane chip G to generate the gel bead-in-emulsion (GEMs). Reverse transcription was primed with an oligonucleotide carrying an Illumina TruSeq R1 read-sequencing primer, a 16 nucleotide 10x cell barcode, a 12 nucleotide UMI, and a 30 nucleotide anchored poly dT sequence. Full length cDNA was amplified from heteroduplex RNA:cDNA using 12 cycles of PCR. The full-length cDNA was cleaned up on SPRIselect beads, and QCed on Qubit and BioAnalyzer. One fourth of the resulting ds cDNA was fragmented and prepared for sample index PCR, with 11 cycles of amplification. After QC, the libraries were pooled and submitted for sequencing on 2 lanes of a 10B 100 flowcell on the Illumina NovaSeqX sequencer, targeting a minimum read depth per cell of 25,000. Sequencing was performed at the UCSF CAT
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
FASTQ files were processed with 10x Genomics’ Cell Ranger analysis pipelines. The read count matrix generated by CellRanger was then analyzed using Seurat v5.0.2. 32288 genes were detected across no ligand (7877 and 7745 cells tested for replicates), GFP pattern (10539 and 11923 cells tested for replicates), mCherry pattern (10671 and 10068 cells tested for replicates), and Dual Pattern (8710 and 16266 cells tested for replicates). Cells that had unique feature counts with at least 700 genes but no more than 7000 genes and cells that had <55% mitochondrial counts were filtered and normalized based on the feature expression and total expression of each cell. The normalized expression data were then used for subsequent analysis.
Principal component analysis was performed after merging replicates and integrating all the conditions. Highly variable genes in each sample after linear transformation and the first 30 PC scores were used for tSNE analysis to cluster the cells into 12 groups (FindNeighbors and FindClusters functions implemented in the Seurat package, dims = 30, resolution = 0.4). The marker genes of each cluster were identified using FindAllMarkers or FindMarkers function with default parameters. Clusters were annotated using signature genes and DAVID pathway analysis to identify fibroblast-, muscle-, or endothelial- like cell types across the different conditions. tSNE clusters that were enriched in proliferation, extracellular matrix, or EGF pathways were identified as fibroblasts. Clusters that were enriched in lineage-specific markers, muscle or angiogenesis pathways, were used to identify muscle- and endothelial- like clusters, respectively.
A pseudobulk method was applied to investigate gene expression among different conditions at the population level. Specifically, the raw gene counts of each sample were extracted after filtering. The counts were then aggregated to the sample level and the expression of genes of interest including transgenes were examined across conditions.
Assembly: mm10
Supplementary files format and content: tar.gz file contains matrix, barcodes, and features
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| Submission date |
Jun 07, 2024 |
| Last update date |
Jun 12, 2024 |
| Contact name |
Song Li |
| E-mail(s) |
songli@ucla.edu
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| Organization name |
UCLA
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| Street address |
404 Westwood Plaza, Eng VI
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| City |
Los Angeles |
| State/province |
California |
| ZIP/Postal code |
90095 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE269381 |
Engineering Programmable Material-To-Cell Pathways Via Synthetic Notch Receptors To Spatially Control Cellular Phenotypes In Multi-Cellular Constructs [snRNA-seq] |
| GSE269404 |
Engineering Programmable Material-To-Cell Pathways Via Synthetic Notch Receptors To Spatially Control Cellular Phenotypes In Multi-Cellular Constructs |
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| Relations |
| BioSample |
SAMN41750506 |
| SRA |
SRX24842150 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8314170_A2_filtered_feature_bc_matrix.tar.gz |
93.0 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
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