title: Illumina HiSeq 1500 paired end sequencing; RNA-seq of CK2alpha-silenced H9-c2 derived myoblasts using siRNA or doxycyclin inducible shRNA.
design: RNA-seq of CK2alpha-silenced H9-c2 derived myoblasts using siRNA or doxycyclin inducible shRNA.
library_construction_protocol: The myoblast cell line H9c-2 derived from embryonic rat myocardium was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultivated at 37°C under a 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Taastrup, Denmark) supplemented with 10% fetal bovine serum (FBS, Biochrom AG, Berlin, Germany). H9c-2 cells were passaged before reaching confluence following guidelines to prevent their differentiation. Cells were transfected with a set of four small interfering RNA duplexes directed against CK2alpha-mRNA, (ON-TARGET plus SMART pools, Dharmacon, CO, USA) using Dharmafect I transfection reagent (Dharmacon) for 72 h according to the manufacturer's recommendations. Control experiments were performed by transfecting cells with scramble-siRNA (Dharmacon).  Total RNA from cells was extracted using Isol-RNA lysis reagent (AH Diagnostics, Aarhus, Denmark) and chloroform followed by precipitation in isopropanol. RNA concentration, purity and integrity were analyzed applying Nanodrop and Agilent 2100 Bioanalyzer RNA 6000 nano kit (Agilent Technologies, Inc., Santa Clara, CA, USA). Only RNA with RIN ≥ 8.0 and a 28s/18s ratio of approx. 1.8 was used for sample preparation. Processing of 500 ng RNA samples for library construction was carried out using the TruSeq Total RNA LT Sample Prep kit, Set A (Illumina) and following the manufacturer's instructions (Illumina TruSeq Stranded Total RNA sample preparation guide). Amplified cDNA libraries were validated in regard to size by Agilent 2100 Bioanalyzer using a DNA 1000 kit from Agilent Technologies and concentration by qPCR using the KaPa Library quantification Kits (KaPa Biosystems, Wilmington, MA, USA).
attributes.tag: Experimental Factor: compound
attributes.value: none
attributes.tag: Experimental Factor: phenotype
attributes.value: wild type phenotype
attributes.tag: Experimental Factor: RNA interference
attributes.value: scrambled siRNA