Extended Data Fig. 2: Rational design of CAT-seq in HeLa cells.
(a) Workflow of in vivo RNA labeling and enrichment. (b) Fluorescence imaging of HeLa cells treated with Ir catalyst (blue) and MitoTracker Red (red). Live cells were performed for 30 min for the catalyst self-localization and then 5 min for MitoTracker Red staining for cellular mitochondria indication. Scale bar, 10 μm. Three biological replicates were repeated with similar results. (c) Immunofluorescent images of HeLa after QMPAB-1 labeling. Green, biotin labeling signal. Red, staining of mitochondrial marker TOMM20. Scale bar: 10 μm. Three biological replicates were repeated with similar results. (d) RT-qPCR analysis of enriched RNA after QMPAB-1 labeling (50 nM photocatalyst and 50 μM QMPAB-1): MTCO1 and MTND1 (mitochondrial mRNAs), GAPDH and ACTNB (cytosolic mRNA markers). Relative abundance is calculated relative to the control without QMPAB-1 probe. Error bars indicate the s.d. calculated from three biological independent biological replicates. n = 3; mean ± s.d. (e) High-resolution images of HeLa after CAT-seq labeling (50 nM photocatalyst and 50 μM QMPAB-2). Green, biotin labeling signal. Red, staining of mitochondrial marker TOMM20. Scale bar: 5 μm. Three biological replicates were repeated with similar results. (f). Chemical structure of catalyst and probe, and immunofluorescent images of HeLa after labeling (50 nM photocatalyst and 50 μM probe). Green, biotin labeling signal. Red, staining of mitochondrial marker TOMM20 or ER marker Calnexin. Blue, Hoechst staining. Scale bar: 10 μm. Three biological replicates were repeated with similar results. (g & h) Immunofluorescent images of HeLa after CAT-seq labeling with different QMPAB-2 treatment time (f) or incubation temperature (g). Green, biotin labeling signal. Red, staining of mitochondrial marker TOMM20. Scale bar: 10 μm. Error bars indicate the s.d. calculated from three independent biological replicates. n = 3; mean ± s.d. P values were calculated by one-sample t-test. Panel a created with BioRender.com.
