Project description:We present RNA-Seq data obtained from HEK293T cells and HEK293T cells with ALKBH5 and FTO demethylases double knockout (HEK293TΔALKBH5ΔFTO), showing m6A-dependent transcriptome alterations.
Project description:RNA-Seq procedure was used for analysis of expression in HEK293T cells. Cells were separately lysed with Trizol reagent (Invotrogen). Total RNA was extracted from cell lysed with Trizol reagent and PureLink RNA Micro Kit (Invitrogen) according to manufacture instruction. RNA quality was checked with BioAnalyser and RNA 6000 Nano Kit (Agilent). Poly(A)+ RNA was purified with Dynabeads® mRNA Purification Kit (Ambion). Illumina library was prepared from poly(A)+RNA with NEBNextNEBNext® Ultra™II RNA Library Prep Kit for Illumina® (NEB) according to manual. Sequencing was performed on HiSeq1500 with 50 bp read length. At least 10 millions of reads were generated for each sample.
Project description:N6-methyladenosine (m6A) is the most abundant, highly dynamic mRNA modification that regulates mRNA splicing, stability, and translation. The m6A epigenetic mark is erased by RNA demethylases ALKBH5 (AlkB Homolog 5) and FTO (Fat mass and obesity-associated protein). The ALKBH5 and FTO RNA demethylases recognize m6A in similar nucleotide contexts. Therefore, these proteins can partially substitute for each other. To assess the impact of total m6A demethylation failure we performed high-throughput sequencing of cytoplasmic RNA from ALKBH5 and FTO double knockout and wild type HEK293T cells. The RNA-Seq libraries were sequenced on Illumina NextSeq 500, trimmed, and mapped to the human genome. The consequent read counting and differential expression analysis in the R environment detected 5871 differentially expressed and 166 alternatively spliced genes comparing double knockout against wild type HEK293T cells. Raw data are deposited in NCBI Gene Expression Omnibus (GEO) repository under GEO accession GSE198050.