MCF10A_CRn_T0
Homo sapiens
Protocols: Inducible Cas9 MCF10A TP53-/- cells were generated for the CRISPRn screen by lentiviral transduction of MCF10A TP53-/- cells with hEF1a-Cas9 (Cat# CAS11229, Dharmacon). Following antibiotic selection with 10 ug/mL blasticidin, 300 million Cas9-expressing cells were infected with a genome wide human lentiviral CRISPR library (RRID:Addgene_67989). The cells were infected with a multiplicity of infection (MOI) 0.3 (to ensure <1 sgRNA per cell), resulting in each sgRNA infecting at least 1,000 cells, a representation that was maintained throughout the experiment. The library contains 90,709 sgRNAs targeting 18,010 genes. Following 2 ug/mL puromycin selection for 72 hours, 1 µg/mL doxycycline was added for 72 hours to induce Cas9 expression. The cell line used for CRISPRi screen was generated by lentiviral transduction of MCF10A TP53-/- cells with lenti-BLAST-dCas9-KRAB (RRID: Addgene_89567) followed by selection with 10 µg/mL blasticidin. 300 million dCas9-KRAB expressing cells were infected at MOI 0.3 with a genome-wide human lentiviral CRISPRi library (RRID: Addgene_84832). The library contains 104,535 sgRNAs targeting 18,905 protein coding genes. In both CRISPRn and CRISPRi screens, cells were collected for an early time point sample of initial library representation (T0) following selection. 100 million CRISPR mutagenized cells were exposed to concentrations that caused a 20% reduction in cell survival (Surviving Fraction 80, SF80) of either 2.5 µM olaparib or 25 nM talazoparib. 25 million cells were seeded into 4X 5 layer flasks and the indicated drug, or DMSO, was added after 24 hours. In total, cells were exposed to drug or DMSO for 14 days (10 population doublings), after which the cells were recovered DNA was extracted from T0 and T1 cell samples using the DNeasy blood and tissue kit (Qiagen), according to the manufacturer's protocol. sgRNA sequences were PCR amplified for sequencing on Illumina sequencing (HiSeq 2500), producing >1,000 short-reads per sgRNA within the library.
ENA-FIRST-PUBLIC
2023-05-31
ENA-LAST-UPDATE
2023-05-31
External Id
SAMEA113428030
INSDC center alias
The Institute of Cancer Research
INSDC center name
The Institute of Cancer Research
INSDC first public
2023-05-31T00:16:04Z
INSDC last update
2023-05-31T00:16:04Z
INSDC status
public
Submitter Id
E-MTAB-12929:MCF10A_CRn_T0
broker name
ArrayExpress
cell_line
MCF-10A
cell_type
epithelial cell
common name
human
genotype
TP53-/-
organism part
breast
sample name
E-MTAB-12929:MCF10A_CRn_T0
scientific_name
Homo sapiens