The CRISPR Screen sequencing raw data

The hBMEC CRISPR/Cas9 library was seeded in eight T225 flasks with 2 × 107 cells each and incubated for 3 days. The hBMECs doubled every 3 days, generating a total of 3.2 × 108 cells, which were divided into four parts, yielding 8 × 107 cells per experimental condition (~1,000× coverage per perturbation in each library). A quarter of the cells were used as the input library and the remaining three quarters for SzM cytotoxicity screens. The cell libraries were treated with purified SzM protein diluted in DMEM (10% FBS) at a final concentration of 50 μg/ml for 48 hr; when ~50% cells were dead, the surviving cells were out grown in fresh DMEM (10% FBS) without SzM protein. These cells were divided into two parts, one part for genomic DNA extraction, and the other for the next round of screening. The same treatment with 50 μg/ml purified SzM protein for 48 hr was used in the 2nd round and the 3rd rounds, using the same protocol outlined above. After the 3rd round, all cells were harvested for genomic DNA extraction. Genomic DNA was obtained from the input hBMEC CRISPR/Cas9 library and after each of the three rounds of screening with SzM protein, the Blood and Cell Culture DNA Maxi Kit. PCR was used to amplify sgRNA sequences and the products were sequenced for amplicon sequencing analysis by the Novegene Co., Ltd.