title: Illumina HiSeq 2500 paired end sequencing; Transcriptomic profiling of HT1080 fibrosarcoma cells of mesenchymal or amoeboid migratory phenotype
design: Transcriptomic profiling of HT1080 fibrosarcoma cells of mesenchymal or amoeboid migratory phenotype
library_construction_protocol: 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.
identifiers.namespace: Charles University in Prague Faculty of Science Department of Cell Biology
identifiers.id: E-MTAB-6823:Sample 11_p