GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM8147537

Query DataSets for GSM8147537

Status

Public on Jul 10, 2024

Title

A375 co-cultured with high ratio of NK cells

Sample type

SRA

Source name

melanoma

Organism

Homo sapiens

Characteristics

tissue: melanoma
cell line: A375
cell type: adherent
genotype: Cas9 overexpression
library treatment: Heidelberg CRISPR library sub-library A
treatment: high amount of human NK cells for 24 hours

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.

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.

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.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

NextSeq 550

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

Submission date

Mar 14, 2024

Last update date

Jul 10, 2024

Contact name

Erica Valentini

Organization name

DKFZ

Street address

Im Neuenheimer Feld 580

City

Heidelberg