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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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4SU-iCLIP of MATR3 from HEK293 cells with or without PTBP1 depletion

ABSTRACT: The aim of the experiment was to compare binding of MATR3 in presence and absence of PTBP1. HEK293T cells were transfected with siRNA targeting PTBP1 and PTBP2 or control sequences and labelled with 4thio-uridine for 8 hours (100µM, crosslinking 2x400mJ/cm2 365nm UV light).

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Homo sapiens

SUBMITTER: Jan Attig

PROVIDER: E-MTAB-6267 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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4SU-iCLIP of PTBP1 from HEK293 cells with or without MATR3 depletion

Project description:The aim of the experiment was to compare binding of PTBP1 in presence and absence of MATR3. HEK293T cells were transfected with siRNA targeting MATR3 or control sequences and labelled with 4thio-uridine for 8 hours (100µM, crosslinking 2x400mJ/cm2 365nm UV light).

2018-05-01 | E-MTAB-6286 | biostudies-arrayexpress

CLIP-Seq of H. sapiens HeLa cells to investigate transcriptome-wide mapping of Matrin3

Project description:Matr3 iCLIP with 4thio-uridine labelling in HeLa cells

2014-12-01 | E-MTAB-3107 | biostudies-arrayexpress

Transcriptome profiling by high throughput sequencing of HeLa cells depleted of MATR3, PTBP1/2 individually or in combination by siRNA.

Project description:We performed RNA-seq experiments on three biological replicates of HeLa cells depleted of MATR3, PTBP1/2, controls, or combined depletion of MATR3/PTBP1/2. Cells were fractionated into cytoplasmic and nuclear RNA. Library preparation was done with the TruSeq stranded RNAseq library kit (Illumina) according to manufacturer’s recommendations; RNA was depleted of rRNA using the RiboZero kit (Epicentre). All libraries were sequenced on Illumina HiSeq2 machines in a single-end manner with a read length of 100 nt.

2018-05-01 | E-MTAB-6204 | biostudies-arrayexpress

Transcriptome 3' end profiling by high throughput sequencing of HeLa cells depleted of MATR3, PTBP1/2 individually or in combination.

Project description:We performed mRNA 3'end sequencing experiments on three biological replicates of HeLa cells depleted of MATR3, PTBP1/2, controls, or combined depletion of MATR3/PTBP1/2. Cells were fractionated into cytoplasmic and nuclear RNAn and only the nuclear RNA was used. Library preparation was done with the QuantSeq library kit (Lexogen) according to manufacturer’s recommendations. Replicates 1 and 2 were prepared with the QuantSeq forward library kit, replicates 3 and 4 with the QuantSeq reverse library kit. All libraries were sequenced on Illumina HiSeq2 machines in a single-end manner with a read length of 100 nt.

2018-05-01 | E-MTAB-6287 | biostudies-arrayexpress

4SU-iCLIP of MATR3 from HEK293 cells with or without PTBP1 depletion

Project description:4SU-iCLIP of MATR3 from HEK293 cells with or without PTBP1 depletion

| PRJEB23752 | ENA

4SU-iCLIP of PTBP1 from HEK293 cells with or without MATR3 depletion

Project description:4SU-iCLIP of PTBP1 from HEK293 cells with or without MATR3 depletion

| PRJEB23801 | ENA

iCLIP of MATR3 from C57BL/6J wildtype mice

Project description:The aim of the experiment was to find binding sites MATR3 in mouse brain tissue. Whole brain was prepped from mice at P0 and replicates are from separate animals.

2018-05-01 | E-MTAB-6283 | biostudies-arrayexpress

CLIP-Seq of H. sapiens HeLa cells to investigate transcriptome-wide mapping of PTB / hnRNP I

Project description:PTB iCLIP-seq in HeLa cells

2014-12-01 | E-MTAB-3108 | biostudies-arrayexpress

Ptpb1 iCLIP in 46C mESCs and mNPCs

Project description:PTBP1 and PTBP2 control alternative splicing programs during neuronal development, but the cellular functions of most PTBP1/2-regulated isoforms remain unknown. We show that PTBP1 guides developmental gene expression by regulating the transcription factor Pbx1. We identify exons that are differentially spliced when mouse embryonic stem cells (ESCs) differentiate into neuronal progenitor cells (NPCs) and neurons, and transition from PTBP1 to PTBP2 expression. We define those exons controlled by PTBP1 in ESCs and NPCs by RNA-seq analysis after PTBP1 depletion and PTBP1 crosslinking-immunoprecipitation. We find that PTBP1 represses Pbx1 exon 7 and the expression of its neuronal isoform Pbx1a in ESC. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of specific neuronal genes including known Pbx1 targets. Thus PTBP1 controls the activity of Pbx1 and suppresses its neuronal transcriptional program prior to differentiation. 46C mESCs and mNPCs were cross-linked at 100 mJ/cm2. Cell pellets were collected and flash-frozen for iCLIP library preparation. Libraries were subjected to 100 single-end RNA-sequencing.

2016-07-03 | E-GEOD-71178 | biostudies-arrayexpress

Modified iCLIP sequencing of HEK293 cells with antibody PTBP1

Project description:For modified iCLIP experiment, 4SU was used for crosslinking as described in published protocol (citation is bellow) and the RNase conditions were optimised to ensure efficient RNase I-dependent fragmentation. In detail, HEK293T cells were grown on 10 cm 2 dishes, incubated for 8 h with 100 M 4SU and crosslinked with 2x 400mJ/cm 2 365nm UV light. Protein A Dynabeads were used for immunoprecipitations (IP). 80 l of beads were washed in iCLIP lysis buffer (50mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate). For the preparation of the cell lysate, 2 million cells were lysed in 1 ml of iCLIP lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) buffer, and the remaining cell pellet was dissolved in 50 L MSB lysis buffer (as above). After the pellet had dissolved, the mixture was diluted with CLIP lysis buffer to 1000 l and an additional centrifugation was performed. Lysates were pooled (2ml total volume) and incubated with 4 U/ml of RNase I and 2 l antiRNase (1/1000, AM2690, Thermo Fisher) at 37C for 3 min, and centrifuged. We took care to prepare the initial dilution of RNase in water, since we found that RNase I gradually loses its activity when diluted in the lysis buffer. 1.5 ml of the supernatant was then added to the beads and incubated at 4C for 4 h. The rest of the protocol was identical to the published protocol (see bellow). Huppertz I, Attig J, D'Ambrogio A, Easton LE, Sibley CR, Sugimoto Y, Tajnik M, Knig J, Ule J: iCLIP: protein-RNA interactions at nucleotide resolution. Methods 2014, 65:274-287.

2016-11-24 | E-MTAB-5027 | biostudies-arrayexpress

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