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PacBio long-read RNA-sequencing to identify differentially expressed genes and repetitive elements between Wild Type RBM4 KO HAP1 cell line samples

ABSTRACT: PacBio long-read RNA-sequencing to identify differentially expressed genes and repetitive elements between Wild Type RBM4 KO HAP1 cell line samples

PROVIDER: PRJNA622498 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11465198_subreads.fastq.gz	Fastqsanger.gz
	SRR11465199_subreads.fastq.gz	Fastqsanger.gz

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PacBio long-read RNA-sequencing to identify differentially expressed genes and repetitive elements between Wild Type RBM4 KO HAP1 cell line samples

Project description:Since short reads from Illumina RNA-seq data are challenging to map to repetitive elements , we wanted to confirm the bulk RNA-seq findings using an orthogonal method, namely, using the long read technology of Pacific Biosciences (PacBio) full-length transcriptome sequencing. This dataset provided around 1.1 (WT) and 1.3 (RBM4 KO) million sequence reads of 2.6 kb average length mapping to the human genome.

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RNA-sequencing experiments to identify differentially expressed genes between Wild Type and RBM4 KO HAP1 cell line samples

Project description:Here, we implemented a computational pipeline to determine the correlation of expression between individual RBPs and ERVs from single-cell or bulk RNA sequencing data. One of our top candidates for an RBP negatively regulating ERV expression was RNA-Binding Motif Protein 4 (RBM4). This set of bulk RNA-sequencing experiments was performed to identify differentially expressed genes and repetitive elements, particularly HERVs, between independent Wild Type and RBM4 KO clones in the KBM-7 derived, near-haploid human cell line, HAP1

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Photoactivatable-Ribonucleotide-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) of the RNA-binding protein (RBP) RBM4 in KBM-7 derived near-haploid human cell line, HAP1.

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Project description:RBM4 is a RNA-binding protein (RBP) able to modulate splicing by promoting exon inclusion and shares an import pathway with other splicing factors in the nucleus. In earlier studies we found that RBM4 interacts and colocalizes with WT1, which has been implicated in 10M-bM-^@M-^S15% of WilmsM-bM-^@M-^Y tumours and more recently in leukemya, is considered to be a tumour suppressor. RBM4, a RBP whose splicing effect is inhibited by the +KTS isoform of the tumour-suppressor/activator WT1, exhibits altered expression in different tumours, and may be essential for proliferation. Moreover, RBM4 binds to a number of RNAs of genes involved in acute myeloid leukaemia and cell cycle control. These results suggest that RBM4 may be involved in alternative splicing, tumorigenesis and particularly leukaemogenesis. To determine the endogenous transcripts that are targeted by RBM4 at the genome-wide level, we decided to perform exon microarray studies. RBM4-specific siRNA has been used for knockdown of RBM4 in HeLa cells. RNA was extracted using a Qiagen RNA extraction kit from untransfected, mock and siRNA-transfected HeLa cells and quality of RNA for microarray analysis was checked using a Bioanalyzer 2100 (Agilent Technologies). Experiments were run in triplicates.

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