Qiamp dna blood midi and maxi kits

CRISPR screens were performed as previously described^{13 (link)}. Lentivirus was produced from the Brunello sgRNA library^{12 (link)} (Addgene 73178) in 293FT cells (Invitrogen) with helper plasmids pPAX2 (Addgene 12260) and pMD2.g (Addgene 12259) in a 4:3:1 ratio in Opti-Mem (Gibco) with Trans-IT 293 T (Mirus) following the manufacturer’s instructions. 293FT supernatants were harvested at 24, 48 and 72 h, pre-cleared by centrifugation at 1000 g for 5 min and concentrated by 40X using Lenti-X concentrator (Takara) following the manufacturer’s instructions. Concentrated Brunello lentiviral library was added to Cas9 MM clones to yield ~30% infection efficiency and maintain ~1 sgRNA per cell with an average of 500 copies per sgRNA in total. Infected MM cells were selected with puromycin 3 days after viral transduction and allowed to grow under selection for another 3 days. At this point, 50 × 10⁶ cells were harvested for the day 0 timepoint and 100 ng/ml of doxycycline and 0.5 μg/ml puromycin was added to at least 50 × 10⁶ cells to induce Cas9 expression, after which a minimum of 50 × 10⁶ cells were passed every other day for 21 days to maintain an average of 500X coverage/sgRNA in the Brunello library. 50 × 10⁶ cells were harvested for the day 21 timepoint. DNA was extracted from Day 0 and 21 cell pellets with QIAmp DNA Blood Midi and Maxi kits (Qiagen).