GSM5530402: Sham+pimobendan biological replicate 3; Mus musculus; RNA-Seq
Under anesthesia with an anesthesia machine (R580, RWD Life Science, China), the mice were weighed and sacrificed by cervical dislocation. We Started below the xiphoid, made a V-shaped thoracotomy, opened the thorax, and exposed the heart. The heart was arrested in diastole with 1% KCl in 0.01M PBS, quickly removed, rinsed with cold PBS, and blotted dried. 1. mRNA Isolation mRNA molecules were purified from total RNA using oligo(dT)-attached magnetic beads. 2. mRNA Fragment mRNA molecules were fragmented into small pieces using fragmentation reagent after reaction a certain period in proper temperture. 3. cDNA Synthesis First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. 4. End Repair, Add A and Adaptor Ligation The synthesized cDNA was subjected to end-repair and then was 3' adenylated. Adapters were ligated to the ends of these 3' adenylated cDNA fragments. 5. PCR This process was to amplify the cDNA fragments with adapters from previous step. PCR products were purified with Ampure XP Beads (AGENCOURT), and dissolved in EB solution. 6. Library Quality Control Library was validated on the Agilent Technologies 2100 bioanalyzer. 7. Circularization 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. 8. Sequencing The library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular. The DNBs were load into the patterned nanoarray and single end 50 (pair end 100/150) bases reads were generated in the way of combinatorial Probe-Anchor Synthesis (cPAS).