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 Control: 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. Thirty million cells were used for genomic DNA isolation to create the unselected control library for sequencing. 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