PTBP1 - Modified iCLIP
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ABSTRACT:
PROVIDER: PRJEB15236 | ENA |
REPOSITORIES: ENA
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Project description:Modified iCLIP sequencing of HEK293 cells with antibody PTBP1
| PRJEB15159 | ENA
Project description:This experiments was performed in HeLa cells according to the iCLIP protocol with the following modifications: no antiRNase was used and the concentration of RNase I was 0.5 U/ml. In iCLIP4, the dephosphorylation step was omitted from the standard protocol. The rest of the protocol was identical to the previously published iCLIP protocol )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-5026 | biostudies-arrayexpress
Project description:The modified iCLIP protocol is based on the previously described protocol (Huppertz et al., 2014) with modifications that enable the definition of readthrough cDNAs. HeLa cells were crosslinked with 0.15mJ/cm2 254nm UV light. Protein G Dynabeads were used for immunoprecipitations (IP). For each IP, 100 l of beads were washed in iCLIP lysis buffer (50mM Tris-HCL pH 7.4, 100mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate), and incubated with the anti-eIF4A3 antibody or polyclonal mAb BB7 serum anti-PTBP1. To prepare the cell lysate, the cells were lysed with 1 mL iCLIP lysis buffer (final concentration 2mg/mL), sonicated (Bioruptor, 5x5 sec on/off), incubated with RNase I (1-2 x10-3 U/mL for eIF4A3) at 37C for 3 min, and centrifuged. After the pellet had dissolved, the mixture was diluted with H2O to 1000 L and an additional centrifugation was performed. The supernatant of the iCLIP lysis buffer (1 mL) and the MSB lysis buffer (1 ml) were combined and added to the antibody-coupled beads, which were then rotated at 4C for 2 h (final urea concentration 175 mM). The beads were then washed with high-salt washing buffer (50mM Tris-HCL pH 7.4, 1M NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate). After the first round of washes, the library was split into 10%, which were radioactively labelled (according to the basic iCLIP protocol), and 90%, which proceed through 3 adapter addition, an additional phosphorylation (0.2 l PNK, 0.4 l cold ATP (1mM), 0.4 l 10x PNK buffer, 3 l water) and a 5 marker ligation (6 l water, 5 l 4X ligation buffer, 2 l RNA ligase, 1 l RNasin, 2 l 5 marker (100 M), 4 l PEG400). The sequence of the 5 marker is CAGUCCGACGAUC, which corresponds to the Illumina short RNA 5 Adapter (RA5), part #15013205; this sequence is not complementary to the primers used for amplification of iCLIP cDNA libraries (Huppertz et al., 2014). We then produced sequence reads of 120 nt using the Illumina HiSeq platform for eIF4A3 iCLIP. The rest of the protocol was carried out as previously described (Huppertz et al., 2014). Huppertz, I., Attig, J., D'Ambrogio, A., Easton, L.E., Sibley, C.R., Sugimoto, Y., Tajnik, M., Konig, J., and Ule, J. (2014). iCLIP: protein-RNA interactions at nucleotide resolution. Methods 65, 274-287.
2016-11-24 | E-MTAB-4000 | biostudies-arrayexpress
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
Project description:We describe an improved individual nucleotide resolution CLIP protocol (iiCLIP), which can be completed within 4 days from UV crosslinking to libraries for sequencing. For benchmarking, we directly compared PTBP1 iiCLIP libraries with the iCLIP2 protocol produced under standardised conditions with 1 million HEK293 cells, and with public eCLIP and iCLIP PTBP1 data. There are 3 PTBP1 iiCLIP libraries, 1 input iiCLIP library and 1 PTBP1 iCLIP2 library produced in this study.
2022-08-01 | E-MTAB-10881 | biostudies-arrayexpress
Project description:We added an oligonucleotide to the 5′ end of RNA fragments —5′ marker— to differentiate between truncated and read–through cDNAs. By applying the protocol to PTBP1 and eIF4A3 iCLIP, we determined that the start sites of read–through cDNAs are enriched in adenosines, while the truncated cDNAs preferentially contained thymidines at their starts.
2018-06-15 | E-MTAB-6927 | biostudies-arrayexpress
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
Project description: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).
2018-05-01 | E-MTAB-6267 | biostudies-arrayexpress
Project description:4SU-iCLIP of MATR3 from HEK293 cells with or without PTBP1 depletion
| PRJEB23752 | ENA