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Status |
Public on Jul 10, 2024 |
Title |
A375 co-cultured with medium ratio of NK cells |
Sample type |
SRA |
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Source name |
melanoma
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Organism |
Homo sapiens |
Characteristics |
tissue: melanoma cell line: A375 cell type: adherent genotype: Cas9 overexpression library treatment: Heidelberg CRISPR library sub-library A treatment: medium amount of human NK cells for 24 hours
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Treatment protocol |
After sgRNA library transduction and puromycin selection, the A375 cells were co-cultured with or without NK cells in an effector-to-target ratio between 1:1 - 1:1.5 which represent medium killing and high killing treatments. Another part of A375 remained untreated and serve as control sample. The co-culture proceeded for 24h followed by NK cell removal. The remaining A375 cell were allowed to expand for 2 days before harvest.
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Growth protocol |
A375 melanoma cells were cultured in RPMI 1640 supplemented with 10 % FCS, 2 mM L-glutamine, and 1 % Penicillin/Streptomycin. Primary NK cells were cultured in SCGM media or in NK MACS media both supplemented with 10% human AB serum, 1% Penicillin/Streptavidin, 2nm L-Glutamine and 400U/ml of IL-2.
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Extracted molecule |
genomic DNA |
Extraction protocol |
gDNA from 50 million melanoma from each sample was isolated using QIAGEN Blood & Cell Culture DNA Maxi Kit. PCR was performed on the purified gDNA to amplify the sgRNA expression cassette. As previously described in Henkel, L., Rauscher, B., Schmitt, B., Winter, J., & Boutros, M. (2020). Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library. BMC biology, 18, 1-21.
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
NextSeq 550 |
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Data processing |
Custom amplicon-seq on the PCR product comprised of sgRNA expression cassette integrated into A375 cells. The raw sequencing data were transformed into a table of the absolute amount of sgRNA read detected from each sample using MAGeCK package Differential gene expression was called from the normalized and log-transformed sgRNA counts from each sample using LIMMA package. Assembly: custom sgRNA library: Heidelberg CRISPR library sub-library A Supplementary files format and content: .fastq.gz: sequence reads of sgRNA expression cassette integrated into A375 genome Library strategy: Amplicon-seq
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Submission date |
Mar 14, 2024 |
Last update date |
Jul 10, 2024 |
Contact name |
Erica Valentini |
Organization name |
DKFZ
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Street address |
Im Neuenheimer Feld 580
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City |
Heidelberg |
ZIP/Postal code |
69120 |
Country |
Germany |
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Platform ID |
GPL21697 |
Series (1) |
GSE261626 |
IFNγ mediates the resistance of tumor cells to distinct NK cell subsets. |
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Relations |
BioSample |
SAMN40462723 |
SRA |
SRX23954574 |
Supplementary data files not provided |
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