GSM3272283: Kdm2a_Knock out_1; Mus musculus; RNA-Seq
Total RNA of cells were extracted by Trizon reagent (CWBIO,CW0580) following the manufacture'sprotocol. For genes expression analysis, total RNA was treated by DNase I (Thermosentific, EN0521) for 30~45min to remove genome DNA. DNase I was inactivated in 70~75 °C for 10min. Next generation sequencing library preparations were constructed according to the manufacturer's protocol (BGISEQ-500 RNA-Seq (Quantification) Library Preparation Protocol). mRNA were fragmented into small pieces using fragmentation reagent. First-strand cDNA was generated using random hexamer-primed reverse transcription , then was followed by a second-strand cDNA synthesis. The synthesized cDNA was subjected to end-repair and then was 3' adenylated. Adaptors were ligated to the ends of these 3' adenylated cDNA fragments. PCR is to amplify the cDNA fragments with adaptors from previous step. PCR products are purified with the SPRI beads, and dissolved in EB solution. The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library . Library was validating on the Agilent Technologies 2100 bioanalyzer. Library was prepared with BGISEQ kit and sequenced on BGISEQ-500 with 50bp read length for 24 million reads.