Rneasy minelute cleanup kit

Differentiation assays for dual reporter mouse embryonic stem cells (ESCs) were conducted as described previously [43 (link)], though compound treatment windows were modified as indicated. Flow cytometry was performed on a BD Accuri C6 or BD LSRFortessa flow cytometer. Synthesis of compounds used in the present study was performed in the Division of Pharmaceutical Chemistry at the University of Helsinki, Pharmatory (Oulu, Finland), Chembridge (San Diego, USA), and Maybridge (Leicestershire, UK) as described [38 (link), 39 (link)]. All-trans retinoic acid (ATRA) and (+)-JQ1 were purchased from a commercial provider (Sigma). Compounds were diluted in DMSO prior to administration (final DMSO concentration 0.1% in medium) and values were normalized to DMSO controls. For characterization of chemically differentiated embryoid bodies (EBs) by qRT-PCR, RNA was isolated from D12 EBs using TRIzol reagent (Thermo Fisher Scientific) and RNeasy MinElute Cleanup kit. qRT-PCR reactions were performed using Taqman gene expression assays (Supplementary Table 1), and values were normalized to a reference gene (Actb). qRT-PCR reactions were performed on a Fluidigim Biomark HD system.

Total RNA was extracted using the extraction reagent TRIzol (cat. no. 15596026, Thermo Fisher Scientific) and further purified using the RNeasy MinElute Cleanup Kit (cat. no. 74204, Qiagen, Valencia, CA, USA). To remove any residual DNA contamination, RNA was digested using DNase I (cat. no. 89254, RNase-free DNase set; Qiagen). RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). First strand cDNA was synthesized from 1 μg total RNA using the iScript™ cDNA synthesis kit (cat. no. 1708890, Bio-Rad, Hercules, CA, USA) in a 25-μL reaction volume according to the manufacturer’s instructions. Gene expression was determined by quantitative real-time PCR using the SsoAdvanced™ Universal SYBR^® Green Supermix (cat. no. 1725271, Bio-Rad, Hercules, CA, USA) in a Mastercycler RealPlex 2 (Eppendorf, Framingham, MA, USA). Primers for CXCL10 (cat. no. qHsaCED0034161) and GAPDH (cat. no. qHsaCED0038674) were obtained from Bio-Rad. The following parameters were used: 2 min at 95 °C, followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C. All samples were normalized using GAPDH housekeeping gene expression. The comparative 2^−ΔΔCT method was used for relative quantitation of the number of transcripts. Non template controls were run in each assay to confirm lack of DNA contamination in the reagents used for amplification.