Positive regulators of IFN lambda signaling
Homo sapiens
Protocols: HAP1 cells were derived from the near-haploid chronic myeloid leukaemia cell line KBM7 as described earlier (Carette et al., 2011). HAP1 cells were cultured in IMDM supplemented with 10% FBS, penicillin-streptomycin and L-glutamine. Cells were mutagenized using a RFP gene-trap virus as described in the article with PMCID number: PMC3111863 Positive regulators of IFN lambda signaling: HAP1 cells were transduced with the vector 5xISRE-eGFP to create an ISG reporter cell line. The reporter cell line was sensitized to IFNλ by overexpressing IFNLR1 (IL28RA). These cells were then sub-cloned to obtain a clonal population prior to mutagenesis. An RFP-gene trap virus was used to create a mutagenized HAP1 reporter library. 100 million mutagenized cells were stimulated with 10 IU/ml IFNλ2. After 24 h, stimulated cells were sorted for GFP-negative cells (approximately 5% of the population) and grown for 3 days. The cells were stimulated again with 10 IU/ml IFNλ2 for 24 h and sorted on GFP-negative cells to enrich for cells that no longer responded to IFN treatment. Thirty million cells of the selected population were used for genomic DNA isolation. Genomic DNA was harvested from 30 million cells using the QIAamp DNA mini kit (Qiagen 51304) Sequencing libraries of genomic regions flanking gene-trap integration sites in control and sorted cell populations were prepared using a linear amplification (LAM) PCR-mediated method described in the article with PMCID number: PMC3111863
ENA first public
2017-07-25
ENA last update
2017-07-11
External Id
SAMEA104158623
INSDC center alias
STANFORD UNIVERSITY
INSDC center name
Stanford University
INSDC first public
2017-07-25T17:01:20Z
INSDC last update
2017-07-11T12:20:18Z
INSDC status
public
Submitter Id
E-MTAB-5866:Positive regulators of IFN lambda signaling
broker name
ArrayExpress
cell line
HAP-1
cell type
haploid cell
common name
human
disease
chronic myelogenous leukemia
genotype
ISRE-GFP reporter and IL28RA overexpression
phenotype
GFP-negative
sample name
E-MTAB-5866:Positive regulators of IFN lambda signaling