Project description:Sequencing was performed to assess the ability of Nanopore direct cDNA and native RNA sequencing to characterise human transcriptomes. Total RNA was extracted from either HAP1 or HEK293 cells, and the polyA+ fraction isolated using oligodT dynabeads. Libraries were prepared using Oxford Nanopore Technologies (ONT) kits according to manufacturers instructions. Samples were then sequenced on ONT R9.4 flow cells to generate fast5 raw reads in the ONT MinKNOW software. Fast5 reads were then base-called using the ONT Albacore software to generate Fastq reads.
Project description:This dataset contains Xdrop followed by oxford nanopore long read sequencing performed in target tRNA gene deletion clones in HAP1 (t72) and HepG2 (t15). By applying de novo assembly based approach to Xdrop-LRS data, we identified Cas9-induced on-target genomic alteration.
Project description:We performed a genome-scale screen for suppressors of interferon stimulated gene (ISG) expression in human haploid cells (HAP1). Ubiquitin specific peptidase 14 (USP14) was a significant hit. In order to validate USP14 as a regulator of ISG expression, we created knockouts of USP14 in HAP1 cells using CRISPR-Cas9 and performed RNA-seq on coding RNA from USP14 KO and WT cells. This data was used to determine if ISGs were upregulated in USP14 KO HAP1 cells.
Project description:We performed a genome-scale screen for suppressors of interferon stimulated gene (ISG) expression in human haploid cells (HAP1). DEAD-box helicase 6 (DDX6) was a significant hit. In order to validate DDX6 as a regulator of ISG expression, we created knockouts of DDX6 in HAP1 cells using CRISPR-Cas9 and performed RNA-seq on coding RNA from DDX6 KO and WT cells. This data was used to determine if ISGs were upregulated in DDX6 KO HAP1 cells.
Project description:Analogous to alternative splicing, alternative polyadenylation (APA) has long been thought to result from competition between proximal and distal polyA sites. By Fractionation-seq, we unexpectedly identified several hundred APA genes where their distal polyA isoforms are retained in chromatin/nuclear matrix and proximal polyA isoforms released into the cytoplasm. Global metabolic PAS-seq and Nanopore long-read RNA-seq provided further evidence that the strong distal polyA sites are first processed and the resulting transcripts are anchored in chromatin/nuclear matrix for further processing at proximal polyA sites and removal of certain slowly spliced introns. By engineering an autocleavable ribozyme between the proximal and distal polyA sites, we demonstrated that the distal polyA isoform is indeed the precursor to the proximal polyA isoform. Therefore, unlike alternative splicing, APA sites are recognized independently, rather than competitively, and in many cases, in a sequential manner. This provides a versatile strategy to regulate gene expression in mammalian cells.