High-throughput sequencing in HNRNPD knock-out SW839 cell lines
As HNRNPD regulates the alternative splicing of hundreds of genes, we sought to investigate whether HNRNPD could regulate the biogenesis of circRNAs. To identify the effect of HNRNPD on circRNAs, we performed circRNA sequencing in HNRNPD knockout and wild-type SW839 cells. In addition, to evaluate the impacts of HNRNPD deficiency on circRNA formation, we captured nascent RNA in SW839 and HNRNPD knock-out SW839 cells by immunoprecipitation of 5-ethyluridine (EU) labeling. Comparative analysis of these data, we found the higher expression level of circRNAs in HNRNPD KO SW839 cells.The HNRNPD could influence the circRNA biogenesis. Overall design: We used CRISPR-Cas9 technology to generate HNRNPD knock-out SW839 cell lines for circRNA and nascent RNA sequencing. we extracted total RNA and digested it with RNase R in HNRNPD knock-out and wild-type SW839 cell lines for circRNA sequencing. For nascent RNA-seq, the cell transcription was terminated with 5,6-dichloro-1-ß-D- ribofuranosylbenzimidazole (DRB, final concentration 0.5 mM) for 3 hours in HNRNPD knock-out and wild-type SW839 cell lines. Subsequently, the culture media was changed with fresh DMEM medium adding 5-ethyluridine (EU, final concentration 0.25 mM) for another 1 hour. Then the nascent RNA was immunoprecipitated with streptavidin-conjugated magnetic beads and extracted with Trizol reagent. The nascent RNAs were to conduct High-throughput sequencing.