4SU-iCLIP of PTBP1 from HEK293 cells with or without MATR3 depletion
ABSTRACT: 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).
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).
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.
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.
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.
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 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 (4x10-3 U/mL for PTB) at 37C for 3 min, and centrifuged. For the PTB iCLIP, the remaining cell pellet was dissolved in 50 L MSB lysis buffer (50mM Tris-HCl, pH 7.4, 100mM NaH2PO4, 7M UREA, 1mM DTT). 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 prepared cDNA libraries with an approximate cDNA size range of 40-140 nucleotides (nt). We then produced sequence reads of 150 nt using the Illumina MiSeq platform for PTBP1.The rest of the protocol was carried out as previously described (Huppertz et al., 2014).
Project description:The vertebrate and neural-specific SR-related protein nSR100/SRRM4 regulates an extensive program of alternative splicing with critical roles in nervous system development. However, the mechanism by which nSR100 controls its target exons is poorly understood. We demonstrate that nSR100-dependent neural exons are associated with a unique configuration of intronic cis-elements that promote rapid switch-like regulation during neurogenesis. A key feature of this configuration is the insertion of specialized intronic enhancers between polypyrimidine tracts and acceptor sites that bind nSR100 to potently activate exon inclusion in neural cells, while weakening 3' splice site recognition and contributing to exon skipping in non-neural cells. nSR100 further operates by forming multiple interactions with early spliceosome components bound proximal to 3' splice sites. These multifaceted interactions achieve dominance over neural exon silencing mediated by the splicing regulator PTBP1. The results thus illuminate a widespread mechanism by which a critical neural exon network is activated during neurogenesis. RNA-Seq was used to obtain mRNA profiles of various N2A and 293T cell lines from human and mouse, respectively, to investigate the roles of nSR100, Ptbp1 and U2af65 in alternative splicing regulation. PAR-iCLIP and iCLIP experiments followed by high throughput sequencing were conducted to obtain RNA binding profiles of nSR100, PTBP1 and U2af65.