PBS-Rag
Cas9 LLC cells were transduced at a MOI of 0.3 with lentivirus produced from the metabolic-focused sgRNA library with at least 1,000-fold coverage (cells per construct) in each infection replicate. Transduced LLC cells were expanded in vitro for 2 weeks and then subcutaneously implanted into Rag1-/- and C57BL/6 mice. The mice were then treated with anti-PD1 or an isotype control on days 7 and 11, and the tumors were harvested on day 15. Genomic DNA from tumors or cells was extracted using a DNA Blood Midi Kit (Qiagen), and sgRNA abundance was determined by NGS after PCR amplification of the sgRNA cassette on an Illumina TruSeq. The manipulation of NGS was conducted in GenePlus (Beijing, China).