Haploid genetic screen for components of MLKL-mediated cell death. Haploid genetic screen for components of MLKL-mediated cell death

One hundred million HAP1 Tet-On-MLKLTE/SD cells were mutagenized with the GFP gene trap retrovirus, as described (PMID: 21623355). The selection experiment was performed by inducing MLKLTE/SD expression with 200 ng/mL DOX over five days and subsequently allowing resistant colonies to grow for four days after withdrawing DOX. Resistant colonies were then pooled together and genomic DNA was extracted from the pool (Qiagen, 51306). Retroviral gene trap insertion sites were amplified using a linear amplification (LAM) PCR protocol as described previously (PMID: 21623355), sequenced using the Genome Analyzer (Illumina).