GSM7734047: WT_GroundState_2; Mus musculus; RNA-Seq
Total RNA was extracted using RNeasy extraction kit (Qiagen) according to manufacturer's instructions including a DNAseI treatment step for 15 min at room temperature (Qiagen) Prior to library construction, purified RNA was quantified by nanodrop and quality assessed by agarose gel electrophoresis and agilent bioanalyser 2100 (to determine RNA integrity and quantity). mRNA was enriched using oligo(dT) beads and then depleted for rRNA using the Ribo-Zero kit (Illumina; according to manufacturer's instructions). The resulting mRNA was converted into sequencing libraries using Illumina protocols performed by Novogene. Briefly, RNA was randomly fragmented by incubation in fragmentation buffer. First strand cDNA synthesis was performed using random hexamer oligonucleotides. Second-strand synthesis was performed using second-strand synthesis buffer (Illumina) containing dNTPs, RNase H and DNA polymerase I. Double stranded cDNA was then taken through terminal repair, A-base ligation, adaptor ligation and size selection steps prior to library amplification. PCR enrichment was performed using illumina barcoding primers and the resulting libraries were quality controlled using a Qubit 2.0, Agilent 2100 bioanalyser and quantitative-PCR.