GSM5739884: MSC-OD-Donor16-Day16-rep3 [thBS_MSC_30]; Homo sapiens; RNA-Seq
GSM5739884
Upon the day of assessment, the medium was discarded using a pipette and 1 mL of RNAzol® RT (Molecular Research Center, Inc., Cincinnati, OH, USA) was added to the wells in question in order to lyse the cells. Repeated pipetting was used to dislodge and lyse the cells completely before moving the resulting lysate to a 2-mL Eppendorf tube. In order to precipitate DNA, protein and polysaccharides 0.4 mL of water was added to the lysate, the mixture shaken for 15 sec and then stored at room temperature (RT) for 15 min. Samples were then centrifuged for 15 min at 12,000 g. Approximately 75% of the resulting supernatant was moved to a new Eppendorf tube. In order to precipitate the total RNA, 1 mL of isopropanol was added to the supernatant collected in the previous step. Samples were then incubated at RT for 10 min before being centrifuged for 10 min at 12,000 g. This collected a small RNA pellet at the bottom of the Eppendorf tube. The supernatant was discarded, and the resulting pellet washed twice by adding 0.5 mL 75% ethanol and centrifuging for two minutes at 5000 g. The pellet was then dissolved in 25 µL of RNase-free water to gain 20 ng/µL concentration for RNA sequencing. cDNA libraries derived from Poly-A mRNA were generated using Illumina's TruSeq RNA v2 Sample Prep Kit. Briefly, Poly-A mRNA was isolated from total RNA samples (0.2–1 μg input) using hybridizaton to Poly-T beads. Poly-A mRNA was fragmented at 94 °C in the presence of divalent cations, followed by first-strand cDNA synthesis using random hexamers and SuperScript IV reverse transcriptase (Invitrogen, Waltham, MA, USA). Following second-strand cDNA synthesis, end repair, the addition of a single A nucleotide, unique dual indexed adaptor ligation (IDT for Illumina), AMPure bead purification, and PCR amplification, the resulting cDNA sequencing libraries were measured on the LabChip GX, diluted to 3 nM, and stored at −20 °C.