title: NextSeq 500 sequencing; MicroRNAs regulate metabolic phenotypes during Multicellular Tumor Spheroids progression
design: MicroRNAs regulate metabolic phenotypes during Multicellular Tumor Spheroids progression
library_construction_protocol: Total RNA extraction Total RNA was isolated from MCF7 monoculture, 6 and 19-day old MCTS by a TRIzol (Invitrogen, CA, USA) adapted protocol. Particular adjustments previous to the TRIzol extraction were made for both culture conditions. In the monoculture, we used a 70-80% confluent culture, then we poured 1 mL of TRIzol directly to the culture dish and homogenized until all cells were detached from the dish. This step was assisted by the use of a scraper. In the 6-day and 19-day old MCTS culture, the MCTS were retrieved by centrifugation and resuspended in 1 mL of TRIzol. The solution was homogenized vigorously until there were no cellular lumps. The subsequent steps described below were used for both culture conditions. We added 10 µL of triton 2% (Merck T8787, St. Louis, USA) and incubated for 10 min. at room temperature. Next, we added 200 µL of chloroform, mixed through inversion and incubated for 5 min. at room temperature. Tubes were centrifuged at 12,000 rpm during 15 min. at 18 ºC. The aqueous phase was retrieved, we added 0.25 µL of glycogen as co-precipitation reagent (ThermoFisher R0561, Waltham, USA) and isopropanol was added in a 1:1 ratio (regarding TRIzol). The mixture was homogenized gently, incubated for 20 min. at −20 ºC and centrifuged at 13,000 rpm during 45 min. at 18 ºC. Finally, the pellet was washed three times with ethanol 75%, dissolved in ultrapure DDW (not treated with DEPC) and stored at −80 °C. For small RNA-Seq, library construction was made by following the manufacturer procedure of CD Small RNA Library Prep Kit