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Single-cell RNA-seq of CRISPRi ADGRG6-kd in iAT2

ABSTRACT: We performed single cell transcriptomic profiling of induced human pluripotent stem cells (iPSCs)-derived type 2 alveolar epithelial cells (iAT2). iPSCs stably expressed CRISPRi (dCas9-KRAB) under the control of doxycyline. iAT2s were transduced with a lentivirus expressing gRNA targeting the transcriptional start site of ADGRG6. Cells were treated with or without doxycyline to intiate CRISPRi-knockdown. Prior to capture, cells were labelled with hashing antibodies (HTO). Cells were captured for 10x Genomics Single cell capture (V3 protocol), and GEX and HTO libraries were sequenced. HTODemux function was used to demultiplex the samples. Knockdown of ADGRG6 caused cells to cluster separately from wild-type cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE223078 | GEO | 2023/09/20

REPOSITORIES: GEO

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Project description:NIPP1, an established interactor of protein phosphatase 1 (PP1), is implicated in PRC2-mediated regulation of gene expression. Here, we explore whether PP1 associated with NIPP1 is involved in NIPP1-mediated regulation of genes. Therefore, we generated Hela Tet-off (HTO) cell lines that stably and inducibly express a Flag-tagged version of wild-type NIPP1 (HTO-NIPP1wt) or mutant NIPP1 (HTO_NIPP1m). The latter mutant lacks the major PP1 binding site by two point mutations in the RVXF-motif, an established PP1 - binding motif. All cell lines were derived from the same parental HeLa Tet-Off (HTO-PT) cell line which expresses only tTA transactivator and is used as a control. The Flag fusions were only expressed in the absence of doxycyline and at levels that were up to twofold higher than that of endogenous NIPP1. We performed a gene expression profiling of the HTO cell lines, using Whole Human Genome Oligo microarrays from Agilent. A Paired SAM analysis identified 1365 genes with an altered expression (P < 0.01) between the Flag-NIPP1 and parental cell lines. Importantly, only 185 genes were differentially expressed (P<0.01) between the Flag-NIPP1m and parental cell lines. Even more strikingly, only 5% of the genes that were affected by the expression of Flag-NIPP1 also showed a significantly different expression in the Flag-NIPP1m cells. A similar small overlap was noted when the analysis was restricted to the 50 genes that were most upregulated or downregulated by the expression of Flag-NIPP1. Finally, scatter plot analysis revealed no significant correlation between the genes that were affected by the expression of Flag-NIPP1 or Flag-NIPP1m. Collectively, these data demonstrate that a moderate increase in the concentration of NIPP1 affects the expression of numerous genes by a mechanism that depends on associated PP1. In total 12 samples were processed. Three different cell lines were analysed: HTO_parental, HTO_NIPP1wt and HTO_NIPP1m. For each cell line 4 replicates were obtained. The HTO_parental cell line is the control cell line. For the HTO_NIPP1wt the 4 replicates were obtained from two replicates of two different transgenic cell lines expressing FlagNIPP1wt (cell line wt n°1 and 2). Each cell line derived from the same parental control cell line.

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Single-cell RNA-seq of CRISPRi ADGRG6-kd in iAT2

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