CRISPR KO screen in U251 cells challenged by Temozolomide.

We generated Cas9-expressing U251 clones by lentiviral transduction (Addgene, ref. 52962-LV) and blasticidine selection. A selected Cas9-expressing U251 clone was expanded, transduced with the Brunello lentiviral library at MOI 0.3 (Addgene, ref. 73178-LV) and selected with puromycine. After expansion to 300 million cells: 100 million were harvested and frozen down, 100 million were treated with 50 uM Temozolomide (TMZ) and 100 million were treated with DMSO (vehicle condition). For the TMZ-treated cells, the medium was changed every 3 days with fresh TMZ. A minimum of 100 million DMSO-treated cells were replated every 3 days in fresh medium. The cells were cultured during 18 days, until the TMZ-treated cells reached the sub-confluence. At this point, DMSO-treated cells and TMZ-treated cells were harvested and frozen down. Genomic DNA were extracted and Illumina libraries were prepared and sequenced.