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TDP-43 iCLIP of cells transfected with UNC13A minigene

ABSTRACT: TDP-43 iCLIP of cells transfected with UNC13A minigene

PROVIDER: PRJEB44081 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR5643240.fastq.gz	Fastqsanger.gz
	ERR5643241.fastq.gz	Fastqsanger.gz
	ERR5643242.fastq.gz	Fastqsanger.gz
	ERR5643243.fastq.gz	Fastqsanger.gz

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TDP-43 iCLIP of cells transfected with UNC13A minigene

Project description:UNC13A contains a cryptic exon which is normally repressed by TDP-43. To better understand the mechanism by which TDP-43 represses this cryptic splicing, and how common SNPs (rs12973192(G) and rs12608932(C)) perturb this regulation, we transfected HEK293T cells with minigenes featuring UNC13A exons 20 and 21, and intron 20. We used two variants of the minigene: one with rs12973192(C) and rs12608932(G) (2x healthy) and the other with rs12973192(G) and rs12973192(C) (2x risk), then performed TDP-43 iCLIP on these cells. We used two replicates for each.

2022-03-24 | E-MTAB-10297 | biostudies-arrayexpress

TDP-43 represses cryptic exon inclusion in FTD/ALS gene UNC13A

Project description:Through splicing analysis of a publicly available RNA-Seq dataset, we discovered TDP-43 represses a cryptic exon splicing event in UNC13A, a gene that had been associated with FTD/ALS through GWA studies. To confirm the sequences of the cryptic exons, we used shRNA to reduce TDP-43 levels in iPSC-derived motor neurons (iPSC-MNs) and by amplicon sequencing the RT-PCR product, we observed the insertion in cells with TDP-43 depletion but not in control shRNA-treated cells. Through sequence alignment, we verified the sequences of the cryptic exons.

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TDP-43 iCLIP of SHSY5Y cells

Project description:TDP-43 is an important RNA binding protein. To better understand its binding targets in human neurons, we performed TDP-43 iCLIP on SHSY5Y cells.

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iCLIP of TDP-43

Project description:iCLIP of TDP-43

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iCLIP of TDP-43

Project description:A stable HEK293 FlpIn T-Rex cells expressing TDP-43 with an N-terminal eGFP-tag was generated that allowed inducible physiological expression of the protein (Ling et al. 2010). Duplicate iCLIP experiments were performed using an antibody targeting eGFP (Abcam ab290). Crosslinked RNA-protein complexes were isolated by immuno-precipitation and cDNAs were generated to allow preparation of Illumina compatible DNA libraries as described in Huppertz et al. (2014).

2017-05-01 | E-MTAB-4733 | biostudies-arrayexpress

TDP-43 iCLIP of SHSY5Y cells

Project description:TDP-43 iCLIP of SHSY5Y cells

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TDP-43 iCLIP in mESC and hESC

Project description:TDP-43 iCLIP in mESC and hESC

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TDP-43 represses cryptic exon inclusion in FTD/ALS gene UNC13A

Project description:TDP-43 represses cryptic exon inclusion in FTD/ALS gene UNC13A

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Mapping hnRNP H binding sites on the RON wildtype minigene via iCLIP.

Project description:We developed a high-throughput mutagenesis screen to comprehensively identify the cis-regulatory elements that control a target splicing event from the MST1R gene that codes for the RON receptor tyrosine kinase. Skipping of alternative exon 11 results in a constitutively active isoform that promotes epithelial to mesenchymal transition and thereby contributes to the invasive phenotype of tumors. We identified the RNA binding protein hnRNP H as an important regulator of RON exon 11 splicing. To map hnRNP H binding sites on the RON minigene, we performed hnRNP H iCLIP with RON wildtype minigene transfected HEK293T cells. iCLIP was performed according to a previously published protocol (PMID: 26463384). The iCLIP libraries were made from two replicates of HEK293T cells at 24 h after RON minigene transfection. The cells were irradiated with 150 mJ/cm2 UV light at 254 nm. For the immunoprecipitation step, we used 7.5 µg of a polyclonal rabbit anti-HNRNPH antibody from Abcam (AB10374). RNase digestion was performed by adding 10 µl of 1/100 diluted RNase I (Ambion) to each sample. We performed the sequencing on an Illumina HiSeq2000 (75-nt single-end reads).

2018-06-27 | E-MTAB-6220 | biostudies-arrayexpress

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