low stringency - uninfected repl A
Genomic DNA was extracted from cells using the QIAmp DNA Mini Kit (Qiagen ref. 51306) or, for the SARS-CoV-2 low stringency screen with the QIAmp DNA Blood maxi kit (Qiagen ref. 51194). In a first PCR step, regions of ~600 bp containing the sgRNA sequence were amplified using NEBNext Ultra II Q5 Master Mix (NEB #M0544S) in 25 amplification cycles. A second PCR of 10 cycles with NEBNext Ultra II Q5 Master Mix was performed with primers containing Illumina adapters and TruSeq indexes. Products were separated by agarose gel electrophoresis and purified with the PureLink Quick Gel Extraction Kit (Thermo Fisher K210012). Samples were then diluted to 24 nM, pooled, and denatured and diluted according to the instructions for single-end sequencing on a MiSeq (Illumina) with a MiSeq-v2 50 cycles or a MiSeq-v3-150 cycles kit (Illumina) and 10% PhiX (Illumina) spike-in.