Oxidative stress sensing by the translation elongation machinery promotes production of detoxifying selenoproteins

The special amino acid selenocysteine plays an essential role in metazoan redox biology and is incorporated into nascent protein chains through the recoding of a UGA termination codon. Using GPX1 and GPX4, two selenoenzymes that protect cells from oxidative stress, as fluorescent reporters we carried out the first genome-wide knockout screen to identify regulators of selenocysteine decoding. Overall design: Two independent clones for each cell line, GPX1, GPX4 and GPX4M, were used for the screen and 1.4x10^8 cells were infected with the Brunello pooled lentiviral CRISPR library (Addgene #73179) in the presence of 5 µg/ml polybrene at a multiplicity of infection between 0.3 to 0.5. Two days after transduction, the cells were selected in medium containing 1 µg/ml puromycin. After 8 days puromycin concentration was lowered to 0.5 µg/ml. After 10 days of selection the top and bottom 0.75% eGFP populations were sorted on a FACS Melody (BD Biosciences). gDNA from sorted pools was isolated by phenol-chloroform extraction. gDNA from 6x10^7 unsorted cells, was isolated with the Masterpure DNA isolation kit (Lucigen). Sequencing libraries were generated from the isolated gDNA through two sequential PCR reactions using the Herculase II DNA polymerase (Agilent). After 18 rounds of initial PCR amplification, 5% of the reaction product was used for a second round of PCR (10cycles) with primers that introduced Illumina sequencing adapters and barcodes. The final PCR products were purified using AMPure XP beads (Beckman Coulter). Libraries were sequenced on a NextSeq2000 (Illumina) with 100bp single-end reads at an average sequencing depth of ~2.2x10^7 reads per sample. sgRNAs sequences were extracted from fastq files through an in-house Galaxy script and normalized reads counts were calculated.