GSM8702679: D4_PTH17_3_Fru; Mus musculus; RNA-Seq
Total RNA was extracted from pTh17 cells or βTH17 cells cultured for 4 days with or without 50 mM fructose using the Direct-zol RNA Microprep Kit (ZYMO Research,R2061). Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-Toligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). 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. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.