Sybr green 1 master mix reagent

Total mRNA was extracted from sorted GFP⁺ cells using MagZol™ Reagent (R4801-03, Magen) according to the manufacturer’s instructions. mRNA purity and quantity were determined with NanoDrop (Thermo Scientific) before qPCR analysis. For qRT-PCR, equal amounts of mRNA samples were reverse transcribed into cDNA using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) Kit (AT341, Transgen). Quantitative real-time PCR was performed on Bio-Rad CFX96 Touch™ Real-Time PCR Detection system using SYBR Green I Master Mix reagent (11203ES03, YEASEN).

Total mRNA was extracted with the MagZolTM Reagent (R4801-03, Magen) following kit directions, and the transcript purity and quantification were evaluated via NanoDrop (Thermo Scientific) prior to qPCR. To conduct RT-qPCR, transcript samples were converted to cDNA with the TransScript All-in-One First-Strand cDNA Synthesis SuperMix (AT341, Transgen), and qPCR was carried out with the SYBR Green I Master Mix reagent (11203ES03, YEASEN) in the Bio-Rad CFX96 TouchTM Real-Time PCR Detection system. The primers used for the qPCR are listed in Table S4. The expression levels of the NKG2DL were normalized using GADPH as the internal control.

Before RNA isolation, BMSCs were incubated with or without cytokines of (50 ng ml^–1 IL-17, 10 ng ml^–1 IFNγ, and 10 ng ml^–1 TNFα) or drugs (100 nM RA and 10 μg ml^–1 BetA), respectively, or jointly for 6 h (Han et al., 2014 (link); Song et al., 2015 (link)). Total mRNA was isolated with MagZol^TM Reagent (R4801-03, Magen) according to the manufacturer’s instruction. mRNA purity and quantity were determined with NanoDrop (Thermo Scientific) before qPCR and RNA-seq analysis. For Real-Time qPCR, cDNA was synthesized from mRNA by using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) Kit (AT341, Transgen). Quantitative Real-Time PCR was performed on Bio-Rad CFX96 Touch^TM Real-Time PCR Detection system with SYBR Green I Master Mix reagent (11203ES03, YEASEN). Sequences of forward and reverse primer pairs are as follows: