Multiplex single-cell chemical genomics reveals the kinase dependence of the response to targeted anti-cancer therapy

Multiplex single-cell chemical genomics reveals the kinase dependence of the response to targeted anti-cancer therapy Single-cell RNA-seq libraries were generated using three-level single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) of untreated or small molecule inhibitor exposed A172, T98G, U87MG, GBM4, GBM8, GSC0131, and GSC0827 glioblastoma brain cancer cells. In a subset of experiments A172, T98G and U87MG engineered to express dCas9-KRAB or dCas9-SunTag for CRISPR/Cas9 mediated gene knockdown or overexpression, respectively, were genetically perturbed for HPRT1, components of the mismatch repair machinery, 522 protein kinases. Different cells and different treatments were hashed and pooled prior to sci-RNA-seq using a nuclear barcoding strategy. This nuclear barcoding strategy relies on the fixation of barcodes containing well-specific oligos that are specific to a given cell type, replicate, or treatment condition. For genetically perturbed experiments, targeted capture and enrichment of gRNA-containing transcripts were performed.

多重单细胞化学基因组学揭示靶向抗癌治疗应答的激酶依赖性。本研究采用三级单细胞组合索引RNA测序（single-cell combinatorial indexing RNA sequencing, sci-RNA-seq），对未经处理或经小分子抑制剂处理的A172、T98G、U87MG、GBM4、GBM8、GSC0131及GSC0827胶质母细胞瘤细胞构建单细胞RNA测序文库。在部分实验中，将经工程化改造以分别表达dCas9-KRAB（用于CRISPR/Cas9介导的基因敲低）或dCas9-SunTag（用于CRISPR/Cas9介导的基因过表达）的A172、T98G及U87MG细胞，针对HPRT1、错配修复机制组分及522种蛋白激酶开展遗传扰动。通过核条形码标记策略，对不同细胞及不同处理组的样本进行标记并混合，随后进行sci-RNA-seq测序。该核条形码标记策略依托带有孔特异性寡核苷酸的条形码，此类寡核苷酸可特异性对应特定细胞类型、重复实验分组或处理条件。针对遗传扰动实验，本研究对携带gRNA的转录本进行了靶向捕获与富集。