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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Aug 29, 2023 |
| Title |
A genetic-metabolic axis for metastatic liver organotropism |
| Organisms |
Homo sapiens; Mus musculus; blank sample |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
To generate CRISPR/Cas9 knockout screen, we used mouse Kinome CRISPR Knockout Library (Brie) (Addgene # 75316) purchased from addgene. The mouse kinase sgRNA pool includes 2,852 sgRNAs targeting 713 kinase genes and 100 control non-targeting sgRNAs. Cas9 expressing HCmel12 melanoma cells were edited for 14 days, and tumor cells were injected for metastasis screening studies. RNA-seq data: HCmel12 melanoma cells and CRISPR knockout HCmel12 melanoma cells - Pip4k2c KO were treated for 4 hours with insulin. Then cells were lysed in RLT buffer and used to perform mRNA profiling.
We performed single-cell RNA-sequencing (scRNA-seq) of eight mice with concurrent liver and lung metastasis. A total of 95,606 cells expressing 19,624 genes were included for downstream analyses.
Bulk RNA-sequencing analysis of 15 human melanoma liver metastasis samples from FFPE sample. Tumor areas were macro-dissected and RNA was extracted using Qiagen kit All Prep DNA/RNA FFPE Kit (Catalog Number 80234), concentration was quantified using Qu-bit and stored at-80 °C until use.
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| Overall design |
In vitro and in vivo CRISPR knockout screen of HCmel12 melanoma cells, and identification of drivers of site-specific metastasis. mRNA profiles following of Parental and Pip4k2c KO HCmel12 melanoma cells with and without insulin treatment for 4hours.
C57/BL6 mice were injected 5x10^5 HCmel12 melanoma cells of indicated genotypes via lateral tail vein and after 21 days mice were euthanized and organs harvested. Around 10,000 cells per channel were loaded on a Chromium controller (10x genomics, Pasadena, CA). Sequencing libraries were prepared using standard parameters, and libraries were sequenced on a NovaSeq with S4 flow cell (Illumina, San Diego, CA) and 2x100 bp paired end reads. For downstream analyses, we include single-cell RNA sequencing of 95,606 cells from 16 samples (MR1-MR16), with matched liver and lung metastasis from HCmel12 mouse melanoma model of indicated genotypes. MR1-MR4 are from HCmel12 melanoma cells with Pip4k2c knockout; MR5-MR10 are from HCmel12 melanoma cells with Pten knockout; MR11-MR16 are from HCmel12 melanoma cells with Pip4k2c and Pten double knockouts.
Bulk RNA-sequencing of 15 independent human melanoma liver metastasis samples were taken from FFPE samples. 5 patients were primary type cutaneous melanoma, 10 were primary type uveal melanoma. Raw counts for each transcript were quantified with kallisto using human reference GRCh38. Raw count data was normalized to transcript per million (TPM).
>>>Submitter states raw data can not be provided for Human samples due to patient privacy concerns<<<
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| Contributor(s) |
Rogava M, Hug C, Izar B |
| Citation(s) |
38286827 |
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| Submission date |
Nov 08, 2021 |
| Last update date |
Jun 28, 2024 |
| Contact name |
Meri Rogava |
| E-mail(s) |
mr4020@cumc.columbia.edu
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| Organization name |
Columbia University
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| Department |
Hematology Oncology
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| Lab |
Izar Lab
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| Street address |
650 W 168th St, New York, NY 10032
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10032 |
| Country |
USA |
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| Platforms (4)
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| GPL17021 |
Illumina HiSeq 2500 (Mus musculus) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
| GPL30314 |
Illumina HiSeq 2500 (blank sample) |
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| Samples (139)
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| Relations |
| BioProject |
PRJNA778715 |
| SRA |
SRP345023 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE188391_RNAseq_normalized_counts.tsv.gz |
1.1 Mb |
(ftp)(http) |
TSV |
| GSE188391_RNAseq_normalized_counts_human.tsv.gz |
1.2 Mb |
(ftp)(http) |
TSV |
| GSE188391_RNAseq_raw_counts.csv.gz |
455.7 Kb |
(ftp)(http) |
CSV |
| GSE188391_RNAseq_raw_counts_human.csv.gz |
469.3 Kb |
(ftp)(http) |
CSV |
| GSE188391_guideRNA_counts.tsv.gz |
290.5 Kb |
(ftp)(http) |
TSV |
| GSE188391_guideRNA_lognorm.tsv.gz |
814.1 Kb |
(ftp)(http) |
TSV |
| GSE188391_sc_barcodes.tsv.gz |
435.7 Kb |
(ftp)(http) |
TSV |
| GSE188391_sc_counts.csv.gz |
449.5 Mb |
(ftp)(http) |
CSV |
| GSE188391_sc_features.tsv.gz |
150.6 Kb |
(ftp)(http) |
TSV |
| GSE188391_sc_matrix.mtx.gz |
397.4 Mb |
(ftp)(http) |
MTX |
| GSE188391_sc_raw_feature_bc_matrix.h5 |
842.1 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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