Single-cell RNA-Sequencing of IMR90 fibroblasts bypassing reprogramming-induced senescence. Homo sapiens

Ectopic expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM) can reprogram somatic cells into induced pluripotent stem cells (iPSCs). These iPSCs are highly similar to embryonic stem cells and can be used for regenerative medicine, drug screening and disease modelling. Despite recent advances, reprogramming is a slow and inefficient process. This suggests that there are several safeguarding mechanisms to counteract cell fate conversion. Cellular senescence is one of these barriers, which is mediated through activation of the tumour suppressors p53/p21CIP1, p15INK4b and p16INK4a. In this study, we have investigated the feasibility of coupling single-cell RNA sequencing (scRNA-Seq) to functional shRNA screening. To this end, we performed scRNA-Seq of OSKM-expressing IMR90 cells infected with shRNAs targeting the top candidates identified in the screen. Overall design: 310 samples: scRNA-Seq libraries of OSKM-expressing IMR90 cells infected individually with shRNAs targeting p53 (40 cells) or shRNAs against the top candidates identified in the screen: p21 (three different shRNAs; 40 cells), MTOR (three different shRNAs; 40 cells), MYOT (two different shRNAs; 40 cells) and UBE2E1 (two different shRNAs; 40 cells). Control samples were: cells expressing OSKM (40 cells) or control vector (40 cells) with empty shRNA vector, 10 positive controls (RNA from IMR90 cells expressing control vector as input) and 10 negative control samples.