GSM6890036: H9c2 cells day 7 without RA, replicate 2; Rattus norvegicus; RNA-Seq
The collection of total RNA extracts was performed following the TRIzol™ reagent protocol instructions (Invitrogen). Add 0.3–0.4 mL of TRIzol™ reagent per 1 × 105-107 cells directly to the culture dish to lyse the cells. The suspension was homogenized pipetting the lysate up and down several times and incubating for 5 min at room temperature to permit the complete dissociation of the nucleoproteins complex. After this time, 0.2 mL of chloroform was added per 1 mL of TRIzol™ reagent used for lysis and incubated for 3 min on ice. Samples were centrifuged for 15 min at 12,000 x g at 4°C, and the aqueous phase that contained the RNA was transferred to a new tube. For the RNA precipitation, we added 0.5 mL of isopropanol to the aqueous phase per 1 mL of TRIzol™ reagent used for lysis, and the sample was incubated for 10 min on ice. The samples were then centrifuged for 10 min at 12,000 x g at 4°C. The supernatant was discarded, and the pellet was resuspended in 1 mL of 75% ethanol per 1 mL of TRIzol™ reagent used for lysis. The sample was vortexed for 5 sec and immediately centrifuged for 5 min at 7500 x g at 4°C; the supernatant was discarded, and the RNA pellet was dried for 10 min on ice. Pellet was resuspended in 50 µL of RNase-free water. Total RNA was quantified using a UV analysis Synergy™ HT Multi-Detection Microplate Reader (Biotek Instruments). To examine RNA integrity and DNA contamination, 0.5 μg of RNA was electrophoretically separated in an agarose gel. Samples were then saved at -80°C. TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer's instructions.