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| Status |
Public on May 22, 2024 |
| Title |
kuramochi_T320_end |
| Sample type |
SRA |
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| Source name |
Cell line
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Cell line
cell line: Kuramochi
cell type: Epithelial
genotype: BRCA2-mutant
treatment: untreated, screen end time point
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| Treatment protocol |
The human metabolic CRISPR/Cas9 knockout library was obtained from the laboratory of Kivanc Birsoy (Birsoy et al. Cell, 2015). Virus was produced by cotransfection of human embryonic kidney–293 (HEK293) cells with lentiviral library pool with packaging vectors (psPAX2 and VSV-G) using PEI Pro (PolyPlus). The titer of lentiviral supernatants was determined by infecting target cells with several amounts of virus in the presence of polybrene (8 μg/ml; Millipore), counting the number of puromycin-resistant infected cells after 3 days of selection. Kuramochi C, T10 and T320 (CD44high/CD24low sorted populations) cells were infected at a multiplicity of infection of ~0.3 and selected with puromycin (4 μg/ml) 48 hours after infection. An initial pool of cells was harvested for genomic DNA extraction (initial point of the screen) as in Birsoy et al. 2015. Remaining cells were cultured for 10 population doublings either off (C, T10, T320) and on olaparib treatment (C in 1 μl and T320 in 40 μl), after which cells were harvested for genomic DNA extraction, constituting the end point of the screen.
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| Growth protocol |
Cells were cultured in RPMI-1640 (ThermoFisher) with 10% Fetal Bovine Serum (FBS), 1% MEM Non-Essential Amino Acids, 1% L-Glutamine 200 mM, 1% Antibiotic-Antimycotic solution 100X and 0.25 U/mL insulin solution (Sigma-Aldrich)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were trypsinized and single-cell suspensions were counted and pellets were frozen for genomic DNA extraction according to Birsoy et al. Cell, 2015.
An initial pool of cells was harvested for genomic DNA extraction as in Birsoy et al. 2015. Remaining cells were cultured for 10 population doublings either off (C, T10, T320) and on olaparib treatment (C in 1 μl and T320 in 40 μl), after which cells were harvested for genomic DNA extraction. sgRNA inserts were PCR-amplified, gel purified, quantified with NEBNext Library Quantification Kit (#E7630S) and sequenced on a Illumina NextSeq 500, generating between 20-40 million paired reads for each library.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
count matrix of sgRNAs from initial and end points of the screen, columns are sample ids
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| Data processing |
Sequencing reads were mapped to the sgRNA library sequences with MAGeCK-count algorithm (Li et al., Genome Biology 2014): mageck count -l Large_metabolic_library_sgRNA_sequence_human.txt --reverse-complement --n output --sample-label sample_labels --fastq fastq_files. The sgRNAs counts for each pair of conditions (initial and end) for treated versus control samples (C) were used as inputs for the MAGeCK-MLE algorithm (Li et al., Genome Biology 2015) to calculate gene scores.
Assembly: hg38/GRCh38
Supplementary files format and content: Large_metabolic_library_sgRNA_sequence_human.tsv, sgRNA sequences
Supplementary files format and content: kuramochi_C_T10_T320_sgrna_counts.tsv, sgRNA counts per sample and condition. Columns are samples and conditions.
Library strategy: CRISPR-Screen
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| Submission date |
Nov 14, 2023 |
| Last update date |
May 22, 2024 |
| Contact name |
Itai Yanai |
| Organization name |
NYU Langone Health
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| Street address |
435 East. 30th St.
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10016 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE247686 |
Cellular adaptation to cancer therapy along a resistance continuum [CRISPR-Screen: Kuramochi] |
| GSE247691 |
Cellular adaptation to cancer therapy along a resistance continuum |
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| Relations |
| BioSample |
SAMN38247387 |
| SRA |
SRX22519680 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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