biostudies-arrayexpressJernej UleHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-13304Arginine-rich mixed charge domains (R-MCDs) contribute to and alter the properties of nuclear speckles. We are interested in how this affects the retention of poly-adenylated mRNAs in the nucleus. This experiment tests how the expression of the R-MCD of PPIG influences the nuclear-cytoplasmic distribution of mRNAs over time. Specifically, a HeLa cell line in which PPIG expression is driven by a doxycycline-inducible promoter was used, and doxycycline was added for 0, 4, 8 or 12 hours. This time course was performed in triplicates. Nuclear and cytoplasmic fractions were collected from the cells and RNA was extracted. 3' end sequencing libraries were produced by fragmenting the RNA and introducing Illumina adapters via a oligo-dT-primed reverse transcription and template switching oligo approach. The data indicate that mRNAs containing long, multivalent GA-rich regions in their coding sequences are more retained in the nucleus over time following expression of the R-MCD. The files provided in this accession have not been trimmed to remove adapters, 3' poly-A sequences, UMIs or G-stretches from TSOs. The data was analysed using nf-core/rna-seq using the following command: nextflow run nf-core/rnaseq \\ --input samplesheet.csv \\ --fasta '/camp/lab/ulej/home/users/farawar/genomes/hs/fasta/GRCh38.primary_assembly.genome.fa' \\ --gtf '/camp/lab/ulej/home/users/farawar/genomes/hs/annotation/gencode.v29.annotation.gtf' \\ --salmon_index '/camp/lab/ulej/home/users/farawar/genomes/hs/salmon_index/salmon_index' \\ --gencode \\ --pseudo_aligner salmon \\ --with_umi \\ --umitools_bc_pattern NNNNN \\ --clip_r1 5 \\ -resume \\ -profile crick \\ --outdir nf-core-results \\ -c extraconfig.config With the extra config file containing: withName: '.*:QUANTIFY_STAR_SALMON:SALMON_QUANT' { ext.args = '--noLengthCorrection' } Therefore removing the first 5 nucleotides as UMI sequences and the following 5 nucleotides as they contain G-stretches from the template-switching oligo.biostudies-arrayexpressNucleic Acid Extraction - RNA extractions were performed from cell pellets using the Maxwell RSC simplyRNA kit (Promega) using a Maxwell RSC Instrument (Promega).Sample Collection - Samples were collected by trypsinising, spinning down, washing in PBS, spinning down and snap freezing on dry ice.Library Construction - RNA was fragmented using magnesium ions and heat, purified, and reverse transcribed using an oligo-dT primer with an Illumina P7 sequence and UMIs. A template switching oligo with the P5 sequence was included in the reaction. The library was then amplified with indexed Illumina i5/i7 primers.Sample Treatment - Pre-warmed doxycycline was added to cells either for either 0, 4, 8 or 12 hours prior to collection.Sequencing - Libraries were sequenced on a NovaSeq 6000 by the Advanced Sequencing Facility at The Francis Crick Institute.Growth Protocol - Cells were grown in DMEM+Glutamax+10% FBS and seeded such that they achieved 70% confluency on the morning of the experiment, with each sample being derived from 2 wells of a 6 well plate.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - All data was processed using the nf-core/rnaseq pipeline as described in the description of the accession.Data Transformation - All data was processed using the nf-core/rnaseq pipeline as described in the description of the accession. Quantification was performed using the STAR-Salmon section of the pipeline. No normalisation for the length of the transcript was applied.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsMaxwell RSC instrumentIllumina NovaSeq 6000RNA-seq of coding RNAHomo sapiensRupert FarawayJernej Ulefalse3' sequencing of nuclear and cytoplasmic fractions following expression of an R-MCDArginine-rich mixed charge domains (R-MCDs) contribute to and alter the properties of nuclear speckles. We are interested in how this affects the retention of poly-adenylated mRNAs in the nucleus. This experiment tests how the expression of the R-MCD of PPIG influences the nuclear-cytoplasmic distribution of mRNAs over time. Specifically, a HeLa cell line in which PPIG expression is driven by a doxycycline-inducible promoter was used, and doxycycline was added for 0, 4, 8 or 12 hours. This time course was performed in triplicates. Nuclear and cytoplasmic fractions were collected from the cells and RNA was extracted. 3' end sequencing libraries were produced by fragmenting the RNA and introducing Illumina adapters via a oligo-dT-primed reverse transcription and template switching oligo approach. The data indicate that mRNAs containing long, multivalent GA-rich regions in their coding sequences are more retained in the nucleus over time following expression of the R-MCD. The files provided in this accession have not been trimmed to remove adapters, 3' poly-A sequences, UMIs or G-stretches from TSOs. The data was analysed using nf-core/rna-seq using the following command: nextflow run nf-core/rnaseq \\ --input samplesheet.csv \\ --fasta '/camp/lab/ulej/home/users/farawar/genomes/hs/fasta/GRCh38.primary_assembly.genome.fa' \\ --gtf '/camp/lab/ulej/home/users/farawar/genomes/hs/annotation/gencode.v29.annotation.gtf' \\ --salmon_index '/camp/lab/ulej/home/users/farawar/genomes/hs/salmon_index/salmon_index' \\ --gencode \\ --pseudo_aligner salmon \\ --with_umi \\ --umitools_bc_pattern NNNNN \\ --clip_r1 5 \\ -resume \\ -profile crick \\ --outdir nf-core-results \\ -c extraconfig.config With the extra config file containing: withName: '.*:QUANTIFY_STAR_SALMON:SALMON_QUANT' { ext.args = '--noLengthCorrection' } Therefore removing the first 5 nucleotides as UMI sequences and the following 5 nucleotides as they contain G-stretches from the template-switching oligo.2025-07-29T00:00:00Z2025-07-30T00:01:26.36Z2023-08-29T10:33:51.468ZE-MTAB-13304ERP150598EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_0003969