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Single-cell RNA Sequencing as a tool for simultaneous shRNA detection and transcriptome analysis in the context of functional shRNA screens

ABSTRACT: 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 screened for shRNAs blunting reprogramming-induced senescence. To evaluate the feasibility of using single-cell RNA Sequencing (scRNA-Seq) in functional screens for simultaneous transcriptome and shRNA identification, we first assessed the accuracy of detecting shRNAs in single cells. To this end we performed a pilot experiment on a pool of OSKM-expressing IMR90 cells infected with the shRNA library.

ORGANISM(S): Homo sapiens

PROVIDER: GSE94979 | GEO | 2018/02/09

REPOSITORIES: GEO

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Coupling shRNA screening with single-cell RNA-Seq identifies mechanisms regulating senescence during reprogramming

Project description: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 screened for shRNAs blunting reprogramming-induced senescence. We integrated single-cell RNA sequencing (scRNA-Seq) with shRNA screening to investigate the mechanism of action of the identified candidates.

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Transcriptome analysis of IMR90 fibroblasts expressing OSKM or control vector and OSKM-induced senescent IMR90 fibroblasts upon p21CIP or mTOR knockdown

Project description: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 screened for shRNAs blunting reprogramming-induced senescence. To investigate the effects of OSKM expression on the transcriptome of human primary IMR90 fibroblasts, we performed RNA-sequencing (RNA-Seq). Cells were transduced with OSKM expression or control vector and RNA was extracted after 14 or 20 days. In addition, to study the transcriptome of cells bypassing OSKM-induced senescence due to p21 or mTOR depletion, we performed RNA-seq of cells transduced with OSKM and shRNA expressing vectors targeting p21 or mTOR for 14 or 20 days.

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Transcriptome analysis of OSKM- or RAS-induced senescent IMR90 fibroblasts treated with Rapamycin.

Project description: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 screened for shRNAs blunting reprogramming-induced senescence. We found that mTOR depletion bypasses OSKM-indced senescence but not RAS-induced senescence. To investigate the differences between the two types of senescence, we performed RNA-sequencing (RNA-Seq). Cells were transduced with OSKM or RAS expression and treated with 2 doses of the mTOR inhibitor Rapaycin for 10 days.

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