High-throughput sequencing in HNRNPD knock-out and knock-in HEK293T 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 and linear RNAs, we performed circRNA and linear RNAs (ployA enrichment RNA and lncRNA) sequencing in HNRNPD knockout and wild-type HEK293T cells. We also inserted 3FHBH (3xFLAG, Histidine, Biotin, and Histidine) into the HNRNPD genome to obtain HEK293T_HNRNPD_3FHBH cells by using CRISPR-Cas9 technology, which was used to ascertain the direct HNRNPD targeting RNA sequence with the help of FLASH sequencing (Fast Ligation of RNA after some sort of Affinity Purification for High-throughput Sequencing). To evaluate the impacts of HNRNPD deficiency on circRNA formation, we captured nascent RNA in HEK293T and HNRNPD knock-out HEK293T cells by immunoprecipitation of 5-ethyluridine (EU) labeling. Comparative analysis of these data, we found the higher propensity of binding to introns of HNRNPD was related to inhibiting circRNA biogenesis. Overall design: We used CRISPR-Cas9 technology to generate HNRNPD knock-out and knock-in HEK293T cell lines. We extracted total RNA and digested it with RNase R in HNRNPD knock-out and wild-type HEK293T cell lines for circRNA sequencing; In the HEK293T_HNRNPD_3FHBH cell, we followed the entire FLASH protocol to collect the RNA that was bound by HNRNPD. Next, the RNA was performed for FLASH sequencing; Culturing to 80% confluence, 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 HEK293T 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 RIP-sequencing.