title: Illumina MiSeq sequencing
design: CRISPR screen readout
library_construction_protocol: Genomic DNA was extracted from 3.6 x 107 pelleted cells per replicate using the DNeasy Blood&Tissue Kit (Qiagen), including an incubation step with 200 µg/ml RNAse A (Qiagen) for 5 min. Concentration of eluted DNA was determined with a Qubit fluorometer (Thermo Fisher Scientific) using the dsDNA HS assay kit (Thermo Fisher Scientific). For amplification of the sgRNA cassette, 25 PCR reactions were performed per replicate, using 1 μg genomic DNA per reaction as input. Amplification was performed as a nested PCR as previously described (citation CLD). In brief, Q5 Hot Start HF polymerase (NEB) was used with the primers SEQ-F1 and SEQ-R1 and the following PCR conditions: 98 °C for 2 min, 25 cycles of 98 °C for 10 s, 62 °C for 15 s, and 72 °C for 30 s, with a final extension at 72 °C for 2 min. The PCR product was cleaned using a QIAquick PCR purification Kit and 5 ng of the eluted DNA was used for a second PCR step with Q5 Hot Start HF polymerase (NEB), primers SEQ-F2 and SEQ-R2, and the following PCR conditions: 98 °C for 2 min, 15 cycles of 98 °C for 10 s, 72 °C for 15 s, and 72 °C for 30 s, and a final extension at 72 °C for 2 min. The product was purified with Agencourt AMPure XP beads at a product-to-beads ratio of 1:1.2. The purified libraries were controlled for correct size using the DNA High Sensitivity Assay on a BioAnalyzer 2100 (Agilent) and then sequenced on a MiSeq (Illumina) by 100-bp single-end sequencing and with the addition of 20 % PhiX Control v3 (Illumina) at a concentration of 8 pM.