GSM8625341: wild-type, replicate 2; Mus musculus; RNA-Seq
Total RNA was extracted using TRIzol (Invitrogen), and its purity was assessed with an Agilent RNA 6000 Nano kit (Agilent). mRNA was purified in two rounds using a Dynabeads mRNA purification kit (Invitrogen). The purified mRNA was fragmented in fragmentation buffer (8 mM Tris, pH 8.2, 50 mM Kac, and 4.5 mM MgAc) and further purified using an RNeasy column. The quality of mRNA isolation and fragmentation was analyzed with an Agilent RNA 6000 Pico kit (Agilent). The fragmented mRNA was used as a template for first-strand cDNA synthesis using random hexamers and the Superscript III First-Strand Synthesis kit (Invitrogen), followed by purification on a Mini Quick Spin column (Roche). Second-strand cDNA synthesis was performed using random hexamers and 200 μM dATP, dCTP, dGTP, and dUTP in the presence of RNase H, Escherichia coli DNA polymerase I, and DNA ligase (Invitrogen). The cDNA samples were purified with a MinElute Reaction Cleanup kit (Qiagen), and >5 ng of cDNA was submitted for library preparation and Illumina deep sequencing. Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) following the manufacturer's instructions. Briefly, DNA was end-repaired, A-tailed, and ligated to Illumina-compatible adapters. The libraries were then amplified by PCR for 12 cycles, purified using AMPure XP beads (Beckman Coulter), and quantified by Qubit (Thermo Fisher). Library quality was assessed using an Agilent Bioanalyzer.