3' sequencing of nuclear and cytoplasmic fractions following expression of an R-MCD

Arginine-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.