biostudies-arrayexpressJernej UleMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-13331In order to investigate the role of SR protein assemblies on GA-multivalent mRNA regions and their relationship to the nuclear speckle. We performed iCLIP of Tra2b, Son-GFP and Srrm2-GFP in mESCs. The GFP-tagged proteins are endogenously tagged and immunoprecipitated using an anti-GFP antibody, while Tra2b was immunoprecipitated using an antibody against the endogenous protein. Srrm2 and Son are very large proteins, but signal was observed at a variety of molecular weights along the membrane, suggesting indirect interactions. We isolated two fractions for each Srrm2 or Son sample: one 'high MW', cut at ~300kDa upwards, and one 'low MW' cut from 40 kDa to 300 kDa. These samples were prepared and sequenced separately. The data provided here has been demultiplexed and trimmed, and therefore should not contain adapter sequences or barcodes. UMI sequences have been moved to the fastq header. Also included are transcriptomic coordinates of deduplicated crosslinks for each sample, using one transcript per gene, as used to plot transcriptomic metaprofiles around multivalent sites.biostudies-arrayexpressGrowth Protocol - mESCs were grown under 2i conditions feeder free, with passaging every 2-3 days and daily media exchanges.Sample Collection - mESCs were grown in 10cm dishes to ~70% confluency, crosslinked with 200mJ/cm2 254nm UV and cell pellets were snap frozen.Nucleic Acid Extraction - 0.4 Units of RNaseI and 4 Units Turbo DNase were added per 1 ml of cell lysate at 1mg/ml protein concentration for RNA fragmentation. Antibodies against GFP (ab290) coupled to magnetic Protein G beads were used to isolate Protein-RNA complexes, and RNA was ligated to a pre-adenylated infra-red labelled IRL3 adaptor. The complexes were then size-separated by SDS-PAGE, blotted onto nitrocellulose and visualised by Odyssey scanning. RNA was released from membrane by proteinase K digestion and recovered by precipitation. cDNA was synthesized with Superscript IV Reverse Transcriptase (Life Technologies) and AMPure XP beads purification (Beckman- Coulter, USA), then circularised using Circligase II (Epicentre) followed by AMPure XP beads purification. After PCR amplification, libraries were size-selected with Ampure beads (if necessary by gel-purification) and quality controlled for sequencing.For the ‘RNase experiment’ we use 4 units Turbo DNase and 0.4U RNase I per 1 ml of lysate at 1mg/ml protein concentration.Sequencing - Samples were sequenced on a HiSeq4000 at the Francis Crick Institute.Library Construction - cDNA was synthesized with Superscript IV Reverse Transcriptase (Life Technologies) and AMPure XP beads purification (Beckman- Coulter, USA), then circularised using Circligase II (Epicentre) followed by AMPure XP beads purification. After PCR amplification, libraries were size-selected with Ampure beads (if necessary by gel-purification) and quality controlled for sequencing.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Sequences were aligned first to a genome containing only ncRNA sequences. Unmapped reads were then aligned uniquely to the mm10 genome using GENCODE M22 as an annotation. STAR was used for both alignments, and for the genomic mapping, STAR also output transcriptomic coordinates of the reads that aligned to spliced transcripts. These alignments were filtered for the longest coding isoform of each protein coding gene.Data Transformation - No normalisation has been applied to the samples provided in this accession.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina HiSeq 4000RNA-seq of coding RNAMus musculusRupert FarawayJernej UlefalseiCLIP of Tra2b, Son-GFP and Srrm2-GFP in mESCsIn order to investigate the role of SR protein assemblies on GA-multivalent mRNA regions and their relationship to the nuclear speckle. We performed iCLIP of Tra2b, Son-GFP and Srrm2-GFP in mESCs. The GFP-tagged proteins are endogenously tagged and immunoprecipitated using an anti-GFP antibody, while Tra2b was immunoprecipitated using an antibody against the endogenous protein. Srrm2 and Son are very large proteins, but signal was observed at a variety of molecular weights along the membrane, suggesting indirect interactions. We isolated two fractions for each Srrm2 or Son sample: one 'high MW', cut at ~300kDa upwards, and one 'low MW' cut from 40 kDa to 300 kDa. These samples were prepared and sequenced separately. The data provided here has been demultiplexed and trimmed, and therefore should not contain adapter sequences or barcodes. UMI sequences have been moved to the fastq header. Also included are transcriptomic coordinates of deduplicated crosslinks for each sample, using one transcript per gene, as used to plot transcriptomic metaprofiles around multivalent sites.2025-09-12T00:00:00Z2025-09-13T04:02:18.555Z2023-09-05T10:01:25.447ZE-MTAB-13331ERP150964EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184