GSM6623994: WbKO_3; Mus musculus; RNA-Seq
Islets were isolated from pancreatic islets of Wtapflox/flox and Wtap-βKO mice at 7 weeks old. One islet sample was combined from three mice. Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany). Three independent biological replicates for each group were used for RNA-seq. mRNA was purified from total RNA by using poly-T oligo-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, then use RNaseH to  degrade the RNA. Second strand cDNA synthesis was subsequently performed using  DNA Polymerase I and dNTP. 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 (Beckman  Coulter, Beverly, USA). Then PCR amplification, the PCR product was purified by  AMPure XP beads, and the library was finally obtained. After the library is qualified, the different libraries are pooling according to the  effective concentration and the target amount of data off the machine, then being  sequenced by the Illumina NovaSeq 6000. The end reading of 150bp pairing is  generated. The basic principle of sequencing is to synthesize and sequence at the same  time (Sequencing by Synthesis). Four fluorescent labeled dNTP, DNA polymerase  and splice primers were added to the sequenced flowcell and amplified. When the  sequence cluster extends the complementary chain, each dNTP labeled by  fluorescence can release the corresponding fluorescence. The sequencer captures the  fluorescence signal and converts the optical signal into the sequencing peak by  computer software, so as to obtain the sequence information of the fragment to be  tested.