Maxwell 16 lev simplyrna cells and tissue kit

Total RNA of cultured PαS cells was extracted using Maxwell 16 LEV simplyRNA Cells and Tissue Kit (Promega). cDNA was synthesized and amplified using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech Laboratories). Data were acquired on an Ion Proton (Thermo Fisher) and analysed using CLC Genomics Workbench (CLC).

Frozen tissue specimens in RNA later® were transferred in 3 ml tubes (Yasui-kiki, Osaka, Japan) and immediately placed in liquid nitrogen. Then, tissue samples were powdered using Multi Beads Shocker® (Yasui-kiki) at 1700 rpm for 10 s. The powdered samples were dissolved in 1 ml TRIzol® reagent (Thermo Fisher Scientific Inc.) and RNA was isolated following the manufacturer’s protocol. Total RNA was extracted using Maxwell® 16 LEV simplyRNA Cells and Tissue Kit (Promega Corporation, Madison, WI, USA) following the manufacturer’s protocol or instruction, and eluted with 50 µl nuclease-free water. The extracted RNA was run on Agilent 2100 Bioanalyzer (Agilent Technologies, Inc. Santa Clara, CA, USA) to determine the quality of RNA. The RNA integrity numbers (RIN) were calculated using the Agilent 2100 Expert Software (Agilent Technologies). The RNA samples with RIN > 7 were used for RNA-seq and qPCR analysis.