Gene regulation by m6A demethylase ALKBH5 in U2OS osteosarcoma. 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.

为鉴定骨肉瘤中ALKBH5调控的靶基因，我们在骨肉瘤细胞系U2OS中敲低ALKBH5的表达，并检测其对U2OS转录组的影响。总体实验设计：将U2OS细胞分别转染阴性对照小干扰RNA（scrambled-siRNA）或ALKBH5小干扰RNA（ALKBH5-siRNAs，Sigma-Aldrich），孵育48小时。转染操作严格依照厂商说明书，采用RNAiMAX转染试剂（Thermofisher）完成。转染48小时后收集细胞，使用RNeasy Mini试剂盒（Qiagen）提取总RNA。全基因组转录组测序方面，我们从转染阴性对照siRNA与ALKBH5-siRNA的U2OS细胞总RNA中构建4个测序文库（敲低组与对照组各设3个生物学重复），文库制备采用TruSeq Stranded mRNA文库制备试剂盒（Illumina Inc.），遵循厂商提供的实验流程。随后使用Illumina HiSeq 3000测序平台（Illumina Inc.），采用50碱基单端读取（50 base-pair single-read, 50SR）测序模块完成测序。