TDP-43 iCLIP of cells transfected with UNC13A minigene
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 4000
ORGANISM(S): Homo sapiens
SUBMITTER: Oscar Wilkins
PROVIDER: E-MTAB-10297 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
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. First, we created a library of mutated minigenes via mutagenic PCR. Importantly, the reverse primer introduced a random barcode sequence which labels the associated mutations. Next, the plasmid library was transfected as a pool and depending on the mutations, the transcripts exhibit changes in alternative splicing. The minigene library and the splicing outcome were analyzed by next-generation sequencing and subsequent integration of the datasets resulted in a map of splicing regulatory sites. The DNA-seq experiment was performed to map the mutations and the associated barcodes in order to identify all minigene variants in the library. For sequencing, we generated five overlapping amplicons of the minigene library using four different forward primers: 5’CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNCTATAGGGAGACCCAAGCTT 3’, 5’CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNGTTCCACTGAAGCCTGAG 3’, 5’CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNAGCTGCCAGCACGAGTTC 3’, 5’CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNGAATCTGAGTGCCCGAGG 3’, and 5’CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNctactggctggtcctcatga 3’, and 5’AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNATAGAATAGGGCCCTCTAGA 3’ as a common reverse primer. After amplification, the PCR products were cleaned using the GeneRead size selection Kit (QIAGEN) according to manufacturer’s instructions. The purified products were first analysed with the TapeStation 2200 capillary gel electrophoresis instrument (Agilent) and then fluorimetrically quantified using a Qubit fluorimeter (Thermo Scientific). Sequencing was carried out on the Illumina MiSeq platform using paired-end reads of 300 nt length and a 10% PhiX spike-in to increase sequence complexity.
2018-06-27 | E-MTAB-6219 | biostudies-arrayexpress
Project description:TDP-43 iCLIP of cells transfected with UNC13A minigene
| PRJEB44081 | ENA
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
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.
2022-02-02 | E-MTAB-11243 | biostudies-arrayexpress
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 with either wildtype or hnRNP H binding site mutant background, we performed hnRNP H iCLIP with RON wildtype or mutant minigene transfected HEK293T cells. iCLIP was performed according to a previously published protocol (PMID: 26463384). The iCLIP libraries were made from two (G331C and G348C) or three replicates (wildtype and G305A) 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 MiSeq or NextSeq500 with 75-nt single-end reads.
2018-06-27 | E-MTAB-6221 | biostudies-arrayexpress
Project description:The occurrence of extensive cryptic splicing following the depletion of nuclear TDP-43 results in impaired neuronal function and degeneration. Here, we show that oligonucleotides containing CA-repeat sequences can mimic TDP-43 pre-mRNA binding and broadly repress cryptic splicing events, reverse protein loss, and restore neuronal activity in TDP-43-depleted neurons. Our results indicate that (CA)n oligonucleotides are potential therapeutic agents to address global mis-splicing in TDP-43 proteinopathies.
2023-12-23 | GSE245513 | GEO
Project description:The occurrence of extensive cryptic splicing following the depletion of nuclear TDP-43 results in impaired neuronal function and degeneration. Here, we show that oligonucleotides containing CA-repeat sequences can mimic TDP-43 pre-mRNA binding and broadly repress cryptic splicing events, reverse protein loss, and restore neuronal activity in TDP-43-depleted neurons. Our results indicate that (CA)n oligonucleotides are potential therapeutic agents to address global mis-splicing in TDP-43 proteinopathies.
2023-12-23 | GSE250255 | GEO
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. First, we created a library of mutated minigenes via mutagenic PCR. Importantly, the reverse primer introduced a random barcode sequence which labels the associated mutations. Next, the plasmid library was transfected as a pool and depending on the mutations, the transcripts exhibit changes in alternative splicing. The minigene library and the splicing outcome were analyzed by next-generation sequencing and subsequent integration of the datasets resulted in a map of splicing regulatory sites. The RNA-seq experiment was performed to quantify the transcript isoforms and sequence their associated barcodes in order to characterize the splicing outcome of the minigene library in HEK293T cells. For preparation of high-throughput RNA sequencing (RNA-seq) libraries, the total RNA obtained from transfected HEK293T cells was enriched for mRNA by performing polyA selection of 20 µg of total RNA using Dynabeads® Oligo (dT)25 beads (Invitrogen) according to the manufacturer’s protocol. Reverse transcription was carried out using 500 ng of enriched mRNA under the abovementioned conditions. To prevent the formation of chimeric amplicons, the libraries were amplified using emulsion PCR (PMID: 16791213), with Phusion DNA Polymerase (NEB) and either cDNA derived from polyA-selected RNA in the case of RNA-seq or plasmid DNA of the minigene library in the case of high-throughput DNA sequencing (DNA-seq). To amplify fragments for RNA-seq, the following primers containing Illumina sequencing adaptors were used: 5′-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNGTTCCACTGAAGCCTGAG-3′ (forward primer) and 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNATAGAATAGGGCCCTCTAGA-3′ (reverse primer). PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter). Purified products were first analysed with the TapeStation 2200 capillary gel electrophoresis instrument (Agilent) and then fluorimetrically quantified using a Qubit fluorimeter (Thermo Scientific). Sequencing was carried out on the Illumina MiSeq platform using paired-end reads of 300 nt length and a 10% PhiX spike-in to increase sequence complexity.
2018-06-27 | E-MTAB-6216 | biostudies-arrayexpress
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.
2021-12-04 | GSE182976 | GEO
Project description:UNC13A contains a novel cryptic exon which is expressed upon TDP-43 knockdown. However, it also features TDP-43 regulated intron retention of a downstream intron. To investigate the correlation of these two events, we performed Nanopore sequencing of amplicons from SHSY5Y cells with inducible TDP-43 knockdown, and FTD patient RNA samples
2022-02-10 | E-MTAB-11244 | biostudies-arrayexpress