High-throughput sequencing in HNRNPD knock-out and knock-in HEK293T cell lines. 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-β-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.

HNRNPD可调控数百个基因的可变剪接，因此我们旨在探究HNRNPD是否能够调控环状RNA（circRNA）的生物发生过程。为明确HNRNPD对环状RNA与线性RNA的影响，我们在HNRNPD敲除与野生型HEK293T细胞中开展了环状RNA测序以及线性RNA（polyA富集RNA与长链非编码RNA（lncRNA））测序。我们还通过CRISPR-Cas9技术将3FHBH（3xFLAG标签、组氨酸标签、生物素标签与组氨酸标签）插入HNRNPD基因组，构建得到HEK293T_HNRNPD_3FHBH细胞系，借助FLASH测序（Fast Ligation of RNA after some sort of Affinity Purification for High-throughput Sequencing）确定HNRNPD直接靶向的RNA序列。为评估HNRNPD缺失对环状RNA形成的影响，我们通过对5-乙基尿苷（EU）标记的新生RNA进行免疫沉淀，分别在HEK293T细胞与HNRNPD敲除HEK293T细胞中捕获新生RNA。通过对上述数据进行比较分析，我们发现HNRNPD与内含子的结合倾向性越高，其对环状RNA生物发生的抑制作用越强。

整体实验设计：我们借助CRISPR-Cas9技术构建了HNRNPD敲除与敲入HEK293T细胞系。在HNRNPD敲除与野生型HEK293T细胞系中，我们提取总RNA并经RNase R酶消化后开展环状RNA测序；在HEK293T_HNRNPD_3FHBH细胞中，我们严格遵循FLASH实验流程收集与HNRNPD结合的RNA，并对其开展FLASH测序。将细胞培养至汇合度80%后，我们在HNRNPD敲除与野生型HEK293T细胞系中加入终浓度为0.5 mM的5,6-二氯-1-β-D-呋喃核糖基苯并咪唑（DRB）处理3小时以终止细胞转录；随后更换添加了终浓度为0.25 mM 5-乙基尿苷（EU）的新鲜DMEM培养基，继续培养1小时。之后使用链霉亲和素偶联磁珠对新生RNA进行免疫沉淀，通过Trizol试剂提取RNA并开展新生RNA免疫沉淀测序（RIP-sequencing）。