Project description:IMR90 ER:RAS cells were infected with 2 different lentiviral pGIPZ shRNAs against AHCY or with an empty vector (EV). We treated IMR90 ER:RAS cells with 4OHT to induce RAS activation or with vehicle (DMSO) as a non-senescent control. RNA was collected 6 days after the start of the experiment once the senescence program has been fully implemented. Our previous experiments showed that knockdown of AHCY prevents senescence. By performing RNAseq, we have unveiled that AHCY knockdown prevents upregulation of a set of genes essential for the onset of senescence and its associated secretory phenotype.
Project description:Purpose: To test the effect of AHCY O-GlcNAcylation on E14.1cell pluripotency and The goals of this study are to compare gene expression profiles of the WT AHCY rescue cells and the T136A AHCY rescue cells. Method:Total RNA was Trizol-extracted and purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEB. cDNA was synthesized using random hexamer primer and RNase H. The library fragments were purified with QiaQuick PCR kits and elution with EB buffer, then terminal repair A-tailing and adapter added were implemented. The clustering of the index-coded samples was performed on a cBot cluster generation system using HiSeq PE Cluster Kit v4-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the libraries were sequenced on an Illumina platform and 150 bp paired-end reads were generated. Results:Significant alterations in gene expression levels were observed, with 1351 genes upregulated and 979 genes downregulated in the T136A AHCY rescue cells
Project description:RNAseq was used to identify host and viral transcriptome changes in methionine restricted Rael cells or Rael cells expressing control, AHCY or MAT2A sgRNAs.