Mirvana mirna isolation kit lysis buffer

hESCs were exposed to all-trans retinoic acid (ATRA in DMSO; MP Biomedicals 02190269.6) at a final concentration of 1 µM in plain TESR-E8 medium supplemented with 1% penicillin/streptomycin (Thermo Fisher Scientific 15140122). Medium was changed daily. Cells were harvested in mirVana miRNA isolation kit lysis buffer (Ambion AM1560) after 0 d (no ATRA) or 1, 3, and 5 d following ATRA induction. Real-time qPCR was used to quantify levels of mRNA expression of four selected genes as previously described in Gingold et al. (2014) (link) and Sagi et al. (2016) (link). First strand cDNA was synthesized from 100 ng of total RNA using SuperScript III RT (Thermo Fisher Scientific 18080093) and random hexamers according to the manufacturer's instructions. qPCR was performed using SYBR Green PCR master mix (Applied Biosystems 10187094) on an Applied Biosystems 7500 real-time PCR system (primer sequences are listed in Supplemental Table S1).

Total RNA of cell lines and tissue samples was extracted using mirVana isolation kit (Ambion AM1560). All tissue samples were kept on dry ice during the initial processing steps. Approximately 30–80 mg of freshly frozen tissue was cut on a cryostat at −20°C and directly disrupted by TissueRuptor II (Qiagen 9002756) with TissueRuptor disposable probes (Qiagen 990890) in mirVana miRNA isolation kit lysis buffer (Ambion AM1560). First, total RNA isolation was performed in an RNase-free environment, followed by on-column RNase-free DNase treatment (Qiagen 79254). Deacylation was then performed by incubating total RNA in 0.1 M Tris-HCL (pH 9.0) and 1 mM EDTA for 30 min at 37°C (Blanco et al. 2014 (link)). After ethanol precipitation, total RNA was resuspended in RNase-free H₂O. Quality and integrity of all RNA samples were measured on a NanodropOne (Thermo Fisher Scientific) and Agilent 2100 bioanalyzer (Agilent Technologies) using an RNA 6000 Nano kit (Agilent Technologies 5067-1511).