Gene regulation by m6A demethylase ALKBH5 in U2OS osteosarcoma

To identify target genes regulated by ALKBH5 in osteosarcoma, we silenced the expression of ALKBH5 in osteosarcoma cell line-U2OS and tested its effect on U2OS transcriptome. Overall design: U2OS cells were were transfected with either scrambled-siRNA or ALKBH5-siRNAs (Sigma-Aldrich) for 48 hours. Transfection was carried out using the RNAiMAX Reagent (Thermofisher) according to the manufacturer's protocol. After 48 hours, cells were harvested and total RNA extraction was done using RNeasy Mini Kit (Qiagen).For whole-genome transcriptome profiling, four libraries (3 replicates each for knockdown and control) were generated from total RNA from scrambled-siRNA- and ALKBH5-siRNA-transfected U2OS cells, using a TruSeq Stranded mRNA Library Preparation kit according to the manufacturer's protocol (Illumina Inc.). Samples were sequenced on the Illumina HiSeq 3000 platform (Illumina Inc.) using the 50 base-pair single-read (50SR) sequencing module.