Project description:SLAM-seq (thiol(SH)-linked alkylation for the metabolic sequencing of RNA) was applied to human primary CD8⁺ T cells subjected to in vitro activation with αCD3/αCD28 beads. To quantify mRNA half-lives and their dynamic changes during CD8⁺ T cell activation, we implemented a pulse-chase design with 24 hours of labelling using 100 µM 4-thiouridine (4SU), replenished every 4 hours with fresh RPMI medium containing 100 µM 4SU. This was followed by uridine washout for 0, 0.5, 1, 3, 6, and 16 hours. Samples were collected from non-activated, Day1-activated, and Day5-activated CD8⁺ T cells at each washout step (2 donors per time point). These datasets provide transcriptome-wide measurements of mRNA half-lives and reveal how mRNA turnover alters upon T cell activation.
Project description:Transcription is regulated by a multitude of activators and repressors, which bind to the RNA polymerase II (Pol II) machinery and modulate its progression. Death-inducer obliterator (DIDO) and PHD finger protein 3 (PHF3) are paralogue proteins that regulate transcription elongation by docking onto phosphorylated serine-2 in the C-terminal domain (CTD) of Pol II through their SPOC domains. Here we show that DIDO3 and PHF3 form a complex that bridges the Pol II elongation machinery with chromatin and RNA processing factors, and tethers Pol II in a phase-separated microenvironment. Their SPOC domains and C-terminal intrinsically disordered regions are critical for transcription regulation. The dataset contains 3' Sequencing of WT, PHF3 KO, and PHF3 dSPOC mutant HEK293 cell line. Cytoplasmic and nuclear fractions were profiled separately.
Project description:The mRNA m6A reader YTHDF2 is overexpressed in a broad spectrum of human acute myeloid leukemias (AML). To study the role of YTHDF2 on mRNA decay rates in leukemia, c-Kit+ cells from foetal livers of Ythdf2fl/fl; Vav-iCre (Ythdf2CKO) and Ythdf2fl/fl (Ythdf2CTL) 14.5 dpc embryos were transduced with Meis1 and Hoxa9 oncogenes and serially re-plated to generate pre-leukemic cells. Medium with 4SU was used for pre-leukemic cells labelling for 12 hours and was later replaced with 4SU-free medium (time 0). Cells were collected immediately after medium change and at 1, 3 and 9 hours for library generation. RNA from Ythdf2CKO (n=3 biological replicates) and Ythdf2CTL (n=3 biological replicates) pre-leukemic cells were used for SLAM-seq library generation.
Project description:We perturbed mRNA degradation machinery in Ascl1-induced neurons (iNeurons) and investigated the change in mRNA half-lives. We performed SLAMseq Metabolic RNA Labeling on Mutant iNeuron line harbouring a ponasteroneA-inducible heterozygous dominant-negative Caf1 (dnCaf1) and on Wild Type iNeurons (WT). The Slamseq technique provided snapshots of mRNA kinetics allowing to estimate mRNA half-lives and assess the effect of mRNA degradation machinery on the level of mRNA stability.