GSM8671598: Co-culture HUVECs and iMSCs Donor 1; Homo sapiens; RNA-Seq
First, 1 mL TRIzol (Invitrogen, Waltham, USA) was added to the samples and incubated for 5 minutes. The gels were pipetted up and down vigorously until completely dissociated in the TRIzol solution and were then frozen at -20 ºC. On the day of isolation, the samples were thawed, mixed with 0.2 mL of chloroform, and thoroughly shaken. This mixture was separated into two phases by centrifugation for 15 minutes at 12,000 g at 4ºC. The aqueous phase was removed and added to 0.5 mL isopropanol to precipitate the RNA. The mixture was incubated for 10 minutes at 4°C, followed by centrifugation at 12,000 g for 10 minutes at 4°C. A white, gel-like RNA pellet was formed at the bottom of the tube and the supernatant was carefully removed using a pipette. To further purify the RNA, the pellet was resuspended in 1 mL of 75% ethanol. The sample was briefly vortexed and then centrifuged at 7,500 g for 5 minutes at 4°C. The supernatant was discarded and the RNA pellet was air-dried for 10-15 minutes until no liquid was left which was then resuspended in RNase-free water. The 3'mRNA-Seq libraries were generated using the QuantSeq 3'mRNA-Seq v2 Library Prep Kit FWD with UDIs (Lexogen, Vienna, Austria), following the protocol provided by the manufacturer.