Illumina MiSeq paired end sequencing; TP53 sequence of human glioblastoma stem cells sorted for p53-Ser15 induction and NS1 virus protein expression upon parvovirus MVMp infection
TP53 sequence of human glioblastoma stem cells sorted for p53-Ser15 induction and NS1 virus protein expression upon parvovirus MVMp infection
Infected GSCs growing as neurospheres in CSC medium (see Growth Protocol section, below) were harvested at 3 days post-infection, in parallel to the respective uninfected controls. The GSCs were selected for the Pp53 and NS1 phenotypes outlined above by fluorescence-activated cell sorting (FACS) as follows. Neurospheres were centrifuged (F15, Falcon) for 5 min at 10.000xg at 4 ºC, dissociated into isolated cells by a mild trypsin-EDTA digestion at room temperature, diluted in PBS and placed on ice. Cells recovered from neurospheres (either Mock or MVM infected) were washed in cold diethylpyrocarbonate-treated (DEPC) phosphate-buffered saline (PBS) prepared with nuclease-free water (Gifco, distilled water DNase/RNase free 10977-035), thoroughly dispersed by flushing through a 0.45 mm needle, fixed with ethanol at -20 °C added dropwise while stirring, and then left to complete fixation for 24 hours at 4 °C. To stain with antibodies, washed cells were permeabilized with PBS-0.1% Tritón, which was also the buffer used in the washes. Antibody staining was performed in autoclaved PBS, 0.1% Triton, 1% gelatin, supplemented with 20 mM Vanadyl Ribonucleoside Complexes (VRC, Sigma) for RNase inhibition, and micro-precipitates removed by filtration through 0.22 m. Incubations with primary (anti-Pp53S15 and anti-NS1; 1/400) and secondary (anti-mouse-Cy5 and anti-rabbit-FITC; 1/500) antibodies were for 16 and 4 hours respectively with rotary agitation at 4 ºC. Finally the cell suspension was washed in PBS-0.1% Triton, clumps removed by filtration (30 ), and concentration adjusted to 106 cell/ml in cold PBS-DEPC, 1% autoclaved gelatin, and 20 mM VRC. Before fluorescence-activated cell sorting (FACS), the equipment circuit was extensively washed with autoclaved PBS-DEPC pre-cooled at 4 °C, and sorting was performed with autoclaved sheath fluid The glioblastoma stem cells (GSCs) were isolated and growth from human brain tumours. Fresh surgical biopsies minced and incubated in 0,1% trypsin and 0,04% DNase (type II, Sigma) in PBS for 45 min at 37 ºC were mechanically dissociated by passing through fire-polished Pasteur pipettes. The cell suspension was filtered through a 40 mm cell strainer (BD), number and viability measured by trypan blue exclusion, and stored frozen in aliquots in liquid nitrogen at this stage. To be grown as Neurospheres, GSCs were seeded at a density of 1x105 viable cells/ml in Cancer Stem Cells proliferation medium (CSC medium) consisting of DMEM:F12 (1:1) supplemented with glutamax (Gibco), 0,5% Albumax I (Gibco), 5 mM Hepes (Gibco), 0,6% Glucose (Sigma), N2 (1x, Invitrogen), 2 mg/ml heparin (Sigma), 20 ng/ml of both epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-2) (Peprotech), aminoacids (L-Ala 44 mM, L-Asn 45 mM, L-Asp 40 mM, L-Glu 40 mM, L-Pro 30 mM)  and gentamicine (0,055 mg/ml) Viral infection, GSC neurospheres grown and fed twice a week with 20% volume of fresh CSC medium until reaching 200-300 mm in size, were dissociated into isolated cells by a mild trypsin-EDTA digestion at room temperature together with mechanically passing through micropipette tips. Cells were washed in CSC and resuspended in the same medium at a density of 1x107 viable cells/ml. The virus was immediately added at a multiplicity of 5-10 PFU/cell and adsorption proceeded under shaking at 37 ºC for 1 h. For the infection we used exclusively purified stocks of the MVMp and MVMi parvovirus strains devoid of empty capsids by two rounds of CsCl centrifugation, dialyzed against phosphate- buffered saline (PBS), and sterile filtered (0.22 m). At the end of the adsorption period cells were washed again and seeded onto P100 dishes at a density of 1x105 viable cells/ml in CSC medium. The FACS-sorted cells were collected on sterile tubes (F15, Falcon) on ice and the TRIzol reagent (Ambion; Invitrogen) was immediately added. Total RNA isolation was performed with chloroform, isopropanol and ethanol, according to the instructions of the manufacturer cDNA was synthetized from 250ng of RNA using the High Capacity cDNA Archive Kit (Applied Biosystems, Thermo Fisher) following the manufacturer´s instructions in a GeneAmp PCR Systems 9700 (Applied Biosystems, Thermo Fisher). From cDNAs we perform a first PCR of 30 cycles with Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs) in the presence of 500nM primers for p53 amplification For each primer pair we optimized the PCR doing a temperature gradient to obtain the optimal annealing temperatures. After the first PCR, a second PCR of 13 cycles was performed with Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs) in the presence of 400nM of primers of the Access Array Barcode Library for Illumina Sequencers (Fluidigm).