RNA-Seq of C2C12 myoblasts
1) There are two methods to treat total RNA. Oligo (dT) magnetic beads are used to select mRNA with polyA tail, or hybridize the rRNAwith DNA probe and digest the DNA/RNA hybrid strand, followed by DNase I reaction to remove DNA probe. Then obtain the target RNA after purification. 2) Fragment the target RNAand reverse transcription to double-strand cDNA (dscDNA) by N6 random primer. 3) End repair the dscDNA to blunt end and phosphate at 5' end, then ligate two blunt end adaptors tothe dscDNA. After ligation, there is a nick between 3' end of insert and adapter. 4)Ligation product is nick translated to intact dsDNAand followed by PCR amplification. 5) Denaturethe PCR product by heat and the single strand DNA is cyclized by splint oligo and DNA ligase. 6) Perform sequencing on prepared library.