GSM7900766: hESC H9, 24hr of no curvature and dual SMAD inhibition after 48hr of dual SMAD inhibition, replicate 2; Homo sapiens; RNA-Seq
RNA was extracted using mirVana™ miRNA Isolation Kit (ThermoFisher). After total RNA purification, the sample was treated with DNase using DNA-Free kit (Ambion). RNA purity was determined by assaying 1 µl of total RNA extract on a NanoDrop8000 spectrophotometer. Total RNA integrity was checked using an Agilent Technologies 2100 Bioanalyzer with an RNA Integrity Number (RIN) value. mRNA sequencing libraries were prepared according to the manufacturer's instructions (Illumina Truseq stranded mRNA library prep kit). mRNA was purified and fragmented from total RNA (1ug) using poly-T oligo-attached magnetic beads using two rounds of purification. Cleaved RNA fragments primed with random hexamers were reverse transcribed into first strand cDNA using reverse transcriptase, random primers, dUTP in place of dTTP. These cDNA fragments then had the addition of a single 'A' base and subsequent ligation of the adapter. The products were purified and enriched with PCR to create the final strand specific cDNA library.