GSM2776477: fetal Normal 6; Homo sapiens; RNA-Seq
Total RNA was extracted using RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Life Technologies, Naerum, Denmark) The Ovation Human FFPE RNA-Seq Multiplex Systems 1-8 and 9-16 (NuGEN, Leek, Netherlands) were used for library preparation from 150-200 ng of total RNA according to the manufacturer’s recommendations with the following modifications: following cDNA synthesis, samples were fragmented to yield fragment sizes of approximately 150 bp using the Covaris E210 sonicator (Covaris Ltd., Brighton, UK). Depending on the initial RNA sizes, the samples were either not fragmented or fragmented for 90, 180 or 360 s. For the final library amplification, a primer amount of 6 μL for each sample and 20 cycles of amplification were used. Following library quality assessment using the Agilent High Sensitivity DNA Kit (Agilent technologies), an extra DNA purification was performed with a 1.4 times overload of Agencourt AMPure XP beads (Beckman Coulter, Copenhagen, Denmark) to get rid of adaptors and primers. Following this, the final library was qualified using the Agilent High Sensitivity DNA kit and quantified using the Qubit dsDNA HS Assay Kit (Life Technologies). DNA was pooled in groups of 9 samples with equal amount of each.