GSM4116258: hi600_2; Mus musculus; RNA-Seq
MEFs were flash frozen in liquid nitrogen and stored at -80 degrees C. Total RNA was extracted using Trizol reagent. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. The mRNA was fragmented randomly by addition of fragmentation buffer. Then first strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Double-stranded cDNA was purified using AMPure XP beads. Remaining overhangs of the purified double-stranded cDNA were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Finally, the final library is retrieved by PCR amplification and purification of PCR products by AMPure XP beads.
GEO Accession
GSM4116258