GSM6731990: INS_1; Mus musculus; RNA-Seq
The mirVana RNA Isolation Kit (Life Technologies) was used to isolate total hepatic or cellular mRNAs.  Agilent 2200 TapeStation Nucleic Acid System was used to determine the RNA integrity number (RIN) and concentration of all RNA samples (Agilent, CA). For the construction of the cDNA library, RNA samples having a RIN greater than 8.5 were employed. rRNA was depleted using hybridization capture to facilitate the identification of protein-coding RNAs. DNase I degrade double-stranded and single-stranded DNA contaminant in RNA samples. mRNA molecules were purified from total RNA using oligo(dT)-attached magnetic beads. mRNA were fragmented into small pieces using fragmentation reagent. First-strand cDNA was generated using random hexamer-primed reverse transcription, then was followed by a second-strand cDNA synthesis. The synthesized cDNA was subjected to end-repair and then was 3' adenylated. Adaptors were  ligated to the ends of these 3' adenylated cDNA fragments. This process is to amplify the cDNA fragments with adaptors from previous step. PCR products are purified with the SPRI beads, and dissolved in EB solution. 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. Library was validating on the Agilent Technologies 2100 bioanalyzer.