Illumina HiSeq 1500 paired end sequencing; RNA-seq of CK2alpha-silenced H9-c2 derived myoblasts using siRNA or doxycyclin inducible shRNA.
RNA-seq of CK2alpha-silenced H9-c2 derived myoblasts using siRNA or doxycyclin inducible shRNA.
The establishment of a cell line expressing shRNA for the inducible down-regulation of CK2α under the control of doxycycline was carried out with the Thermo Scientific SMARTchoice inducible shRNA kit following the manufacturer's instructions (Thermo Scientific, Rockford, IL, USA). The SMARTchoice inducible shRNA vector consists of an inducible promoter with tetracycline response element (PTRE3G), activated by the Tet-On 3G protein in the presence of doxycycline (Dox). The SMARTvector contains a turbo-GFP (tGFP) reporter gene and a universal scaffold carrying shRNA directed against the CK2α sequence GAA.TTA.GAT.CCA.CGT.TTCA. Initially, general transduction conditions and the functional titer of viral particles were optimized, according to the manufacturer's recommendations. Cells were treated with optimal viral titer in transduction medium containing 10% serum and 10 μg/ml polybrene for 20 h followed by addition of normal growth medium for further 24 h. Transduced cells were selected with 0.3 μg/ml puromycin for three days, re-seeded as single cells and allowed to grow until each clone contained 30 to 40 cells. The clones were analyzed by Western blot for the efficient down-regulation of CK2α following treatment with 1 μg/ml doxycycline (Dox). Analysis of green fluorescence-emitting cells was done on a FACSCalibur flow cytometer and data acquisition was carried out with Cell Quest Pro Analysis software (BD 18 Biosciences, Franklin Lakes, New Jersey, USA). Cell pictures were taken with a Leica DMRBE fluorescence microscope equipped with a DFC 420C camera and Leica Application Suite V 3.3.0 software (Leica Microsystem, Wetzler, Germany). 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 treated with 1 μg/ml doxycycline (Dox) or double-distilled sterile water as negative control 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).
Experimental Factor: compound
none
Experimental Factor: phenotype
wild type phenotype
Experimental Factor: RNA interference
CK2alpha doxycyclin induced knockdown by siRNA