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

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

PROVIDER: PRJEB23752 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR2210802.fastq.gz	Fastqsanger.gz
	ERR2210803.fastq.gz	Fastqsanger.gz
	ERR2210804.fastq.gz	Fastqsanger.gz

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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

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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).

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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.

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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.

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