title: Illumina Genome Analyzer II sequencing; Haploid genome-wide screens for essential components and negative regulators of interferon lambda signaling in human cells
design: Haploid genome-wide screens for essential components and negative regulators of interferon lambda signaling in human cells
library_construction_protocol: 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 Negative regulators of FN 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. To sort for cells that displayed constitutive activation of the reporter in the absence of IFN, 100 million mutagenized, unstimulated, cells were sorted for the top 5% GFP positive by FACS and grown over a period of 4 days. Cells were then re-sorted for the top 5% GFP positive to enrich for cells displaying constitutive reporter activation. 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