GSM3559974: WT-1; Homo sapiens; RNA-Seq
Total RNA of cells were extracted by RNAiso plus reagent (TaKaRa) 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. 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.
GEO Accession
GSM3559974