Secondary siRNA screen (5-ethynyl uridine RNA metabolic labelling)

Secondary image-based genetic screen targeting 463 genes in human HeLa cells using RNA interference (siRNA), as described in Berry et al, 2021. Cells incubated with 5-ethynyl uridine (5-EU) for 30min before fixation. Nascent RNA detected by click reaction with AlexaFluor488-azide. Other stains: DAPI (DNA), proliferating cell nuclear antigen (PCNA), succinimidyl ester AlexaFluor647 (total protein). Cells fixed 72h after siRNA transfection. Imaged at 20X magnification with two experimental replicates per condition. Further description available in Berry et al., 2022. Single-cell features provided as background-subtracted mean and sum intensity values for the nucleus, cytoplasm, or whole-cell, as appropriate, for each fluorescence channel. Cell-cycle (G1/S/G2) annotation using a classifier trained using PCNA and DAPI features on EdU-incubated cells, as described in the manuscript. Data cleanup and correction applied as described in the manuscript. Summaries provided on a per-well basis.本数据集为一项基于成像的二次遗传筛选，以人类HeLa细胞为研究模型，采用RNA干扰（RNA interference, siRNA）技术靶向463个基因，相关研究细节参见Berry等人2021年的研究成果。细胞在固定前先经5-乙炔基尿苷（5-ethynyl uridine, 5-EU）孵育30分钟；新生RNA通过与AlexaFluor488-叠氮（AlexaFluor488-azide）的点击化学反应实现标记检测。其余染色标记包括：DAPI（标记DNA）、增殖细胞核抗原（proliferating cell nuclear antigen, PCNA）、经琥珀酰亚胺酯标记的AlexaFluor647（succinimidyl ester AlexaFluor647，用于标记总蛋白）。细胞于siRNA转染72小时后完成固定。成像采用20倍放大倍率，每个实验条件设置两次实验重复。更详细的实验描述可参见Berry等人2022年的研究成果。单细胞特征以背景校正后的平均强度与总强度值形式提供，针对每个荧光通道，分别根据实际情况提取细胞核、细胞质或全细胞的对应数值。细胞周期（G1/S/G2）注释通过预训练分类器完成，该分类器基于经EdU孵育细胞的PCNA与DAPI特征训练得到，相关细节详见研究手稿。数据清理与校正流程均按照研究手稿中的描述执行。数据集以每孔为单位提供汇总统计结果。