10x 3' scRNA-seq analysis on Jurkat cells post TCR acivation in CRISPR/Cas9 screening

By performing 10x 3' scRNA-seq on Jurkat cells post TCR acivation in CRISPR/Cas9 screening, we want to investigate the compatibility of the A/G mixed capture sequence on multiple single cell RNA-seq platforms. By mimicking the adenylated endogenous mRNA, gRNA transcripts could be directly captured by poly(dT) primer during the reverse transcription, and serve as perturbation index in high identification rate. Overall design: In this study, we provided a framework of direct 'genotyping' for single cell CRISPR screen, by incorporating a capture sequence into the gRNA scaffold. With that, genotype, transcriptome and phenotype can be analyzed simultaneously. We used an A/G mixed capture sequence to mimic the poly(A) tail of pol II transcripts and facilitate the direct annealing of gRNA with poly(dT) RT primer that is widely used in versatile scRNA-seq platforms.

本研究通过在CRISPR/Cas9筛选体系中对T细胞受体（T cell receptor, TCR）激活后的Jurkat细胞开展10× 3'端单细胞RNA测序（single cell RNA-seq, scRNA-seq），旨在探究A/G混合捕获序列在多款单细胞RNA测序平台上的兼容性。通过模拟腺苷酸化的内源信使RNA（messenger RNA, mRNA），向导RNA（guide RNA, gRNA）的转录本可在反转录（reverse transcription, RT）过程中直接被寡聚dT引物捕获，并作为扰动指数实现高识别效率。实验整体设计：本研究通过将捕获序列整合至gRNA骨架中，构建了一套适用于单细胞CRISPR筛选的直接「基因分型」研究框架。依托该框架，可同时实现基因型、转录组与表型的联合分析。本研究采用A/G混合捕获序列，模拟RNA聚合酶II（RNA polymerase II, pol II）转录本的poly(A)尾，以促进gRNA与广泛应用于各类单细胞RNA测序平台的寡聚dT RT引物的直接退火结合。