Egm 2 bullet kit tissue culture medium

Reagents were obtained and used as received. DNA templates and primers were obtained from Integrated DNA Technologies. The RNA oligonucleotide competitors and precursor RNAs, obtained from Dharmacon, were 2′-ACE-protected (where ACE is 2′-bis(acetoxyethoxy)-methyl ether) and were deprotected according to the manufacturer’s protocol. All RNAs were desalted using PD10 Sephadex (GE Healthcare) columns by first pre-equilibrating the column with 25 ml water, then loading the RNA and eluting with 10 ml water, collecting 1 ml fractions. Autoradiography images were obtained using a Typhoon 9410 variable mode imager (GE Healthcare) and quantified using Quantity One (Bio-Rad) software. All UV-vis measurements for RNA quantification were obtained using a Beckman Coulter DU800 UV-vis spectrophotometer by heating the RNA to 90 °C and measuring the absorption at 260 nm. Complementary DNA (cDNA) samples were quantified using an Agilent Technologies 2100 Bioanalyzer (Model: G1939A) and an Invitrogen Qubit 2.0 Fluorimeter. Subsequent sequencing was carried out on a Life Technologies Ion Proton sequencer with at least 200-fold coverage. EGM-2 Bullet Kit tissue culture medium obtained from Lonza (CC-3162) was used to grow HUVECs directly. All cells were cultured at 37°C in 5% CO₂ in 100-mm-diameter dishes unless stated otherwise.