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( A ) RT-qPCR detection of chimeric RNA UBA1-CDK16 in myeloid cells, NK cells, CD4 + T cells, CD8 + T cells, and B cells sorted from human PBMCs ( n = 2). ( B ) RT-qPCR detection of chimeric RNA UBA1-CDK16 on days 0, 3, and 5 of CD34 + cells myeloid differentiation ( n = 2). ( C ) Experimental timeline for shRNA transduction of expanded CD34 + cells followed by myeloid differentiation. ( D ) The effect of UBA1-CDK16 knockdown by shUC1 and shUC2. Short hairpin green fluorescent protein (shGFP) was used as negative control ( n = 2). ( E ) The fluorescent intensity distribution of CD11b in CD34 + cells undergo myeloid differentiation upon UBA1-CDK16 knockdown. Gated-out single cells were used for fluorescence-activated cell sorting (FACS) analysis. ( F ) CD11b mRNA expression detected by RT-qPCR. RNA samples were extracted from day 5 of myeloid differentiated cells. ( n = 3). ( G ) Uniform manifold approximation and projection (UMAP) projection of single-cell transcriptomes from shUC1 and shGFP samples from a female donor, clustered into major blood cell types. Cell types were annotated on the basis of canonical marker gene expression. MEPs, megakaryocyte-erythroid progenitors.

Journal: Science Advances

Article Title: UBA1-CDK16 : A female-specific chimeric RNA emerging through evolution and involved in immune regulation

doi: 10.1126/sciadv.adz9784

Figure Lengend Snippet: ( A ) RT-qPCR detection of chimeric RNA UBA1-CDK16 in myeloid cells, NK cells, CD4 + T cells, CD8 + T cells, and B cells sorted from human PBMCs ( n = 2). ( B ) RT-qPCR detection of chimeric RNA UBA1-CDK16 on days 0, 3, and 5 of CD34 + cells myeloid differentiation ( n = 2). ( C ) Experimental timeline for shRNA transduction of expanded CD34 + cells followed by myeloid differentiation. ( D ) The effect of UBA1-CDK16 knockdown by shUC1 and shUC2. Short hairpin green fluorescent protein (shGFP) was used as negative control ( n = 2). ( E ) The fluorescent intensity distribution of CD11b in CD34 + cells undergo myeloid differentiation upon UBA1-CDK16 knockdown. Gated-out single cells were used for fluorescence-activated cell sorting (FACS) analysis. ( F ) CD11b mRNA expression detected by RT-qPCR. RNA samples were extracted from day 5 of myeloid differentiated cells. ( n = 3). ( G ) Uniform manifold approximation and projection (UMAP) projection of single-cell transcriptomes from shUC1 and shGFP samples from a female donor, clustered into major blood cell types. Cell types were annotated on the basis of canonical marker gene expression. MEPs, megakaryocyte-erythroid progenitors.

Article Snippet: Antibodies were added to stain surface markers for 30 min at 4°C (CD3 phycoerythrin (PE), Life Technologies; CD8 fluorescein isothiocyanate (FITC), Life Technologies; CD11b AF700, Life Technologies CD19 PerCP-Cy5.5, BioLegend; C33 BV605, BioLegend; CD56 BV711, BD).

Techniques: Quantitative RT-PCR, shRNA, Transduction, Knockdown, Negative Control, Fluorescence, FACS, Expressing, Single Cell, Marker, Gene Expression