GSM1356837: 60% 3243G 143B; Homo sapiens; RNA-Seq
Total RNA was extracted with 1ml of Trizol directly applied to the culture dish. Total RNA was depleted of cytosolic rRNA (RiboMinus, Life Technologies #10837-08). RNA was quality checked and quantified on a Bioanalyzer 2100, RNA 6000 Nano kit (Eukaryote Total RNA Nano, Agilent Techonolgies) and Qubit 2.0 fluofometer RNA assay kit (Molecular Probes #Q32852). Five hundred ng of RNA was used for cDNA library preparation using (Ambion, Total RNA-Seq kit #4445374) according to manufacturer's instructions. Samples were barcoded such that they could be pooled and run on the same sequencing lanes. RNA from all eight experimental groups (transmicotochondrial cybrid cell lines 0, 20, 30, 50, 60, 90 and 100 % 3243G heteroplasmy plus the Rho0 parental cell line) were sequenced on the ABI SOLiD 5500 platform. Barcoded triplicates were sequenced on the same slide in different lanes. All sequencing was performed using paired-end chemistry of 50 (forward) x 35 (reverse) base pairs.