RNA-seq of wildtype and PIEZO1 knockout mice livers to investigate lipid and glucose regulation by the endothelial force sensor PIEZO1
RNA-seq of wildtype and PIEZO1 knockout mice livers to investigate lipid and glucose regulation by the endothelial force sensor PIEZO1
Liver of 12-14 week-old male mice was used and tissue was mechanically separated using forceps, further cut in smaller pieces and incubated at 37 Â°C for 50 min, in a MACSMix Tube Rotator to provide continuous agitation, along with 0.1% Collagenase II (17101- 015, Gibco, Waltham, MA) and Dispase Solution (17105-041,Gibe). Total RNA was prepared from tissues using TriZol reagent (Sigma). RNA sequencing was performed by Novogene, UK. After the QC procedures, mRNA from eukaryotic organisms 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.