GSM9755767: LF_1; Mus musculus; RNA-Seq
RNA was harvested using standard extraction methods, followed by strict quality control of the RNA samples, primarily using the Agilent 2100 Bioanalyzer: precise assessment of RNA integrity. A total amount of 1 μg RNA per sample was used as input material for the RNA samplepreparations.Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads.Fragmentation was carried out using divalent cations under elevated temperature in FirstStrand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using randomhexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNAsynthesis was subsequently performed using DNA Polymerase I and RNase H. Remainingoverhangs were converted into blunt ends via exonuclease/polymerase activities. Afteradenylation of 3' ends of DNA fragments, Adaptor with hairpin loop structure were ligated toprepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp inlength, the library fragments were purified with AMPure XP system (Beckman Coulter,Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase,Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPureXP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.