Illumina HiSeq 2500 paired end sequencing; Transcriptomic profiling of HT1080 fibrosarcoma cells of mesenchymal or amoeboid migratory phenotype
Transcriptomic profiling of HT1080 fibrosarcoma cells of mesenchymal or amoeboid migratory phenotype
One million HT1080 cells were cultured in a 500 μl three-dimensional gel made of rat tail collagen (Serva, 1.0 mg/ml) in DMEM with 1% fetal bovine serum for 48 hours in a 24-well plate. Gels from two wells were transferred into one 2 ml tube and homogenized with Tissue Tearor (BioSpec Products) in 600 μl of RNA extraction solution (60% v/v water-saturated phenol, 3.25 M guanidine thiocyanate, 400 mM sodium acetate buffer pH 4.0, 0.4% w/v N-lauroylsarcosine, 160 mM 2-mercaptoethanol) plus 100 μl of 6.1 M sodium chloride. Before the experiments, HT1080 cells were expanded in DMEM supplemented with 10% fetal bovine serum and 50 μg/ml gentamicin (all from Sigma). HT1080 cells bearing the doxycycline-inducible EGFP-RhoA G14V construct were treated with 250 ng/ml doxycycline. HT1080 cells bearing the LifeAct-mCherry construct were treated with 1 μM dasatinib (final concentration) in DMSO or equivalent volume of DMSO only (0.1% final concentration). 200 μl of chlorophorm was added to approx. 1 ml of the lysate and the mixture was vortexed vigorously for 10 seconds. After 30-minute centrifugation at 18,000 g at 4°C, the polar upper phase was transferred to a new tube, volume adjusted to 800 μl with RNase-free water, RNA was precipitated with 600 μl of isopropanol and recovered by centrifugation at 18,000 g for 30 minutes. The RNA pellet was washed three times with 600 μl of 75% ethanol and air-dried. Next, the RNA was treated with DNAse I to remove possible genomic DNA contamination. To this end, the pellet was directly dissolved in 100 μl of solution containing 4 units of DNAse I (Thermo Fisher Scientific) in manufacturer-provided reaction buffer and incubated at 37°C for 30 minutes. After that, the RNA was re-purified with RNeasy Protect Mini Kit (Qiagen) according to the manufacturer's instructions and eluted in 50 μl of RNase-free water.  Stranded, Illumina HiSeq-compatible library was constructed with ScriptSeq Complete (Human/Mouse/Rat) library preparation kit (Epicentre) according to the manufacturer's instructions.
Charles University in Prague Faculty of Science Department of Cell Biology
E-MTAB-6823:Sample 5_p
Experimental Factor: compound
none
Experimental Factor: genotype
doxycycline-mediated expression of EGFP-RhoA G14V
Experimental Factor: phenotype
mesenchymal