title: GSM9054766: siRNA MYO6 2; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted from three replicates of MYO6 KD (siRNA duplex (5′GGUUUAGGUGUUAAUGAAGtt-3′) (Ambion)) and scrambled siRNA (using control siRNA, Qiagen, 1027280) in MCF-7 cells. Ice cold TRIzol reagent was added to each cell culture dish and homogenised. The mixture was incubated at room temperature for 10 minutes. Chloroform was then added to the mixture and incubated at room temperature for 5 minutes. The samples were centrifuged at 8,000 xg. The top, aqueous layer was collected and isopropanol was added. The mixture was then centrifuged at 12, 000 xg for 30 minutes and the supernatant discarded. The pellet was washed in 75% (v/v) ethanol and centrifuged at 7, 500 xg for 5 minutes. The pellet was air dried and resuspended in RNAse-free H2O. RNA concentration and quality was then assessed by measuring absorbance at 260 nm and A260/A280 ratio. RNA samples were stored at -80°C. The following procedures were performed by  Glasgow Polyomics. The RNA-seq libraries were prepared using Poly-A selection. Resulting libraries concentration, size distribution and quality were assessed on a Qubit fluorometer and on an Agilent 2100 bioanalyzer. Paired-end sequencing was then performed on an (2x75 bp) on a HiSeq sequencer  (Glasgow Polyomics). The following procedures were performed by Glasgow Polyomics. The RNA-seq libraries were prepared using Poly-A selection. Resulting libraries concentration, size distribution and quality were assessed on a Qubit fluorometer and on an Agilent 2100 bioanalyzer.