GSM5928166: Liver macrophages Normal Diet 18 weeks M670; Mus musculus; RNA-Seq
Cells were lyzed in Trizol for 10 minutes (min) at room temperater (RT). Then 200μl chloroform were added. Votex to mix well and stand at RT for 15 min. After 10 min centrifuge at 12000rpm and 4oC, upper clear layer (350μl) with RNA inside was collected. Later steps were performed with a QIAprep Spin Miniprep Kit according to the instrucions provided. After the QC procedures, mRNA was enriched using oligo(dT) beads.  First, the mRNA was fragmented randomly by adding fragmentation buffer, then the cDNA was synthesized by using mRNA template and random hexamer primers, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I were 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 was completed through size selection and PCR enrichment.