GSM4932940: Undifferentiated/proliferative CAD rep1; Mus musculus; RNA-Seq
Total RNAs were extracted from undifferentiated CAD cells (Pro 1-3) and Dex-differentiated cells (5 days, Dex 1-3) using the TRIzol reagent (reference number 15596026, lot number 262308, Ambion, Life technologies Corporation, USA), according to the manufacturer's instructions. Three independent samples were collected from both experimental groups. RNA concentration and purification were preliminary examined using a NanodropTM 2000 spectrophotometer (Thermo Fisher Scientific, USA). Quality of the submitted total RNAs were evaluated using a Bioanalyzer (Agilent, USA). All mRNA libraries were constructed using 500 ng of input total RNA, as outlined by the QIAseq Standard mRNA Select kit (Qiagen, USA). QIAseq Beads were used to separate the cDNA from the reaction mix. Subsequently, indexing adapters were ligated to the cDNA, and the cDNA libraries were enriched using 12 cycles, as recommended by the Qiagen CleanStart library amplification protocol. Library quality assurance was conducted using a Bioanalyzer and fluorometer (DeNovix, USA). Next, a library size as recommended by Illumina for this type of library was observed to demonstrate a successful library preparation. Finally, all libraries were combined equally at 10 nM and further diluted to 1.8 pM. The sequencing step was performed on NextSeq with a 75 base-pair end read length.