title: Illumina HiSeq 2000 sequencing; Cell size gene expression in Drosophila Kc167 cells
design: Cell size gene expression in Drosophila Kc167 cells
library_construction_protocol: Kc167 cells were grown in Schneiders medium containing 10% FBS. Cells were transfected with dsRNAs targeting each gene using FugeneHD transfecton reagent. After 4 days, aliquot of cells were analysed by flow cytometry and remaining cells were collected for RNA extraction. Cells were lysed with QIAzol Lysis Reagent (Qiagen). After chloroform addition, total was precipitated from the aqueous phase with 1/10 volume of 3M sodium acetate, pH 5.0 and one volume of isopropanol. 15 ug total was fragmented for 3 min at 70 degreesC in fragmentation buffer (Ambion). Fragmentation was terminated by cooling of the sample on ice and addition of EDTA to 17 mM final concentration. 2.5 ul fragmented was then mixed with 0.5 ul 100 mM MgCl2 and diluted to 30 ul with RNAse-free water.
identifiers.namespace: University of Dundee
identifiers.id: E-MTAB-2651:pAbp2.3seq