GSM6451113: Mouse RNA-seq - ISO 1hour rep3; Mus musculus; RNA-Seq
After 1 hour, 1 day, or 3 days, mice were euthanatized following anesthetization with an overdose of pentobarbital. The heart tissue was collected for further determination. A total amount of 1 μg RNA was used as input material for the RNA sample preparations. mRNA was purified from total RNA by using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase, then use RNaseH to degrade the RNA. Second strand cDNA synthesis was subsequently performed using dNTP with DNA Polymerase I system. After adenylation of 3' ends of DNA fragments, the adaptor with hairpin loop structure was ligated for hybridization. To select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). After PCR amplification, the PCR product was purified by AMPure XP beads, and the library was finally obtained. To ensure the quality of the library, the library was initially quantified by Qubit2.0 Fluorometer, then diluted to 1.5ng/ul, and the insert size of the library is detected by Agilent 2100 bioanalyzer. After insert size meets the expectation, qRT-PCR is used to accurately quantify the effective concentration of the library (the effective concentration of the library is higher than that of 2nM) to ensure the quality of the library.