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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 04, 2023 |
| Title |
KT, replicate 2, in vivo RNA-seq |
| Sample type |
SRA |
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| Source name |
lung
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| Organism |
Mus musculus |
| Characteristics |
tissue: lung
genotype: KT
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| Extracted molecule |
total RNA |
| Extraction protocol |
KT, KFT, and KPT mice were transduced with Ad5-SPC-Cre and harvested for cell sorting at 10 weeks post-transduction. Non-transduced lungs were used as a control. Briefly, lungs were perfused with 10 mL of ice-cold PBS and placed in a sterile dish on ice. Lungs were finely minced with sterile scissors and razor blades and resuspended in 6 mL digestion media (RPMI with 2.5 mg of Liberase TL (Sigma-Aldrich) and 25 µg/mL DNase (Sigma Aldrich)). Lungs were rotated for 45 minutes at 37ºC. Tubes were briefly cooled on ice, and then samples were passed through 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).
For RNA-sequencing, 1.5x105 FACS sorted tumor cells were harvested, pelleted, and flash frozen in liquid directly after sorting. Once all samples were collected, RNA was harvested using the RNeasy Micro Kit (Qiagen). Quality and concentration of RNA was determined using a Bioanalyzer. cDNA libraries were constructed using the Trio RNA-seq library preparation kit (NuGEN) from samples with high quality RNA according to the manufacturer’s instructions. Samples were sequenced on a HiSeq4000 (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
RNA-seq
counts_AK2_KT
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| Data processing |
RNA-seq reads were aligned to the mouse genome (mm10) using HISAT2 (version 2.0.5, https://github.com/DaehwanKimLab/hisat2), sorted based on genomic location and indexed using Samtools (version 1.3.1, https://github.com/samtools/samtools), and counted and mapped to each gene using HTSeq-Count (version 0.6.1, https://github.com/simon-anders/htseq). Differentially expressed genes (DEGs) were identified using DESeq2 with a cutoff of a p-adjusted value of < 0.05 (version 1.24.0, https://github.com/mikelove/DESeq2).
Assembly: mm10
Supplementary files format and content: counts file, each column is a sample
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| Submission date |
May 04, 2023 |
| Last update date |
May 04, 2023 |
| Contact name |
Laura D Attardi |
| E-mail(s) |
[email protected]
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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 |
GPL21103 |
| Series (2) |
| GSE231679 |
p53 governs an alveolar type 1 differentiation program in lung cancer suppression [in_vivo_RNA_seq] |
| GSE231681 |
p53 governs an alveolar type 1 differentiation program in lung cancer suppression |
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| Relations |
| BioSample |
SAMN34591368 |
| SRA |
SRX20229300 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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