GSM7057359: S2G_TTF1.0y_miNSC_4F1_P8_R1; Mus musculus; RNA-Seq
Total RNA was extracted using Trizol reagent (Invitrogen, #15596018) following the user guide quantified using a Nanodrop One spectrophotometer (Thermo Scientific). mRNA is enriched using oligo(dT) beads. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina), dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, a ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment. ffective concentration of library was determined by Q-PCR. Finally, the qualified libraries are fed into Illumina sequencers after pooling according to its effective concentration and expected data volume.