GSM7732630: SK-MEL-2_CRISPRi_day7_replicate2; Homo sapiens; OTHER
Genomic DNAs of the two group cells were extracted by FastPure Cell/Tissue DNA Isolation Mini Kit (Vazyme, DC102) for PCR amplification (Vazyme, N616), and the amplified products were subject to library construction using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, ND607) according to the kit's instructions and then performed the next-generation sequence on a HiSeq X Ten platform. The CRISPRi screen was independently performed for two times. We first comprehensively curated essential genes associated with melanoma cell growth and drug resistance from published CRISPR/shRNA screening studies. Then, we selected HRR-associated enhancers receiving both H3K27ac and H3K4me1 signals in A375 cells within 1Mb region of each melanoma essential genes. These enhancer regions were further narrowed with chromatin accessibility signal based on ATAC-seq peaks. For the remaining enhancer core regions, we divided each region into several bins consisting of 100bp and scored all eligible sgRNAs in each bin using FlashFry. By selecting the best sgRNA in each bin, the intersected tiling strategy can yield a sgRNA library for all selected enhancers, and a total of 1,000 unmatched sgRNAs were added into the library as negative control. The sgRNA sequences were synthesized by Synbio Technologies and cloned into lentiGuide-Puro vector (Addgene, 52963) in the form of homologous recombination to construct the plasmid library, and then packaged into the lentivirus library using the pMD2.G (Addgene, 12259) and psPAX2 (Addgene, 12260) packaging plasmids for subsequent screening.