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| Status |
Public on May 04, 2023 |
| Title |
SpcPT, replicate 1, scRNAseq |
| Sample type |
SRA |
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| Source name |
lung
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| Organism |
Mus musculus |
| Characteristics |
tissue: lung
genotype: SpcPT
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| Extracted molecule |
total RNA |
| Extraction protocol |
SpcT and SpcPT mice treated as described above. Due to mouse colony limitations, one SpcT mouse was Sftpc-CreERT2;p53+/+;Rosa26Confetti (Brainbow 2.1)85. However, RFP is only expressed after Cre treatment, as in SpcT mice, and only red RFP+ cells were sorted from this mouse in an identical manner as the replicate SpcT mouse. 96 hours after BHT treatment, lungs were harvested and perfused with 10 mL of ice-cold PBS and placed in a sterile dish on ice. Lungs were minced with sterile scissors and resuspended in 6 mL digestion media (RPMI with 0.083 U/mL of collagenase type IV (Sigma-Aldrich), 0.6 U/mL dispase ii (Sigma-Aldrich), and 25 µg/mL DNase (Sigma Aldrich)). Lungs were rotated for 30 minutes at 37ºC. Tubes were briefly cooled on ice, and then samples were passed through a 70 µM filter followed by a 40 µM filter. 5 mL of FACS buffer (10% FBS, 2 mM EDTA, 25 µg/mL DNase in DPBS) was added and tubes were spun for 5 minutes at 300g. Cells were resuspended in ACK lysing buffer (Gibco), incubated for 1 minute on ice, and quenched with 8 mL of FACS buffer. Cells were washed 2x with FACS buffer, resuspended with biotinylated primary antibodies (CD45, BioLegend 103104 30-F11; CD31, BioLegend 102404 390; F4/80, BioLegend 123106 BM8; Ter119, BioLegend 116204 TER-119, 1:800 in FACS buffer) and incubated for 20 minutes on ice. After washing 2x, cells were resuspended in streptavidin-APC secondary antibody (BioLegend 405207, 1:800) for 20 minutes on ice. Cells were washed 2x and resuspended in FACS buffer with 1 µg/mL DAPI, filtered through a 40 µM filter, and sorted using a Sony SH800S cell sorter. Data were analyzed using the default Sony SH800S software and FCS Express (Version 7, De Novo Software).
Single cell suspensions were prepared from mice treated with BHT as described above. Libraries were prepared using the 10x Genomics Single Cell 3’ Library v3.1 kit according to the manufacturer’s protocols. An input of 10,000 cells were added to each 10x channel. Libraries were sequenced on an Illumina NovaSeq6000.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10x Genomics
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| Data processing |
Analyses were performed in R (version 4.0.3) using various R packages and Python (version 3.6) unless otherwise noted using the Stanford SCG Informatics Cluster. FASTQ files were processed using cellranger count (10x genomics). This pipeline aligns reads to the mm10 mouse reference genome, performs barcode error correction, PCR duplicate marking, barcode counting, and UMI counting to produce expression count matrices. The gene expression matrices were loaded into R and processed with Seurat (version 4.0.1, https://github.com/satijalab/seurat) for downstream analyses.
Assembly: mm10
Supplementary files format and content: filtered feature matrix CellRanger output
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| Submission date |
May 04, 2023 |
| Last update date |
May 04, 2023 |
| Contact name |
Laura D Attardi |
| E-mail(s) |
attardi@stanford.edu
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| Organization name |
Stanford University
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| Lab |
Attardi
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| Street address |
269 Campus Drive
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE231675 |
p53 governs an alveolar type 1 differentiation program in lung cancer suppression [scRNA-Seq] |
| GSE231681 |
p53 governs an alveolar type 1 differentiation program in lung cancer suppression |
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| Relations |
| BioSample |
SAMN34591333 |
| SRA |
SRX20229339 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7295751_SpcPT_filtered_feature_bc_matrix.h5 |
18.6 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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