title: DMSO_Vehicle_1
organism: Rattus norvegicus
description: Protocols: Primary rat vascular smooth muscle cells were isolated from 40-50 days old male Wistar rats weighing 170-250 g. Animals were sacrificed by decapitation and fast bleeding. The thoracic aorta was excised and placed in modified Krebs-Ringer solution (120 mM NaCl (Duchefa Biochemie); 4.7 mM KCl (Duchefa Biochemie); 1.8 mM CaCl2 (Duchefa Biochemie); 0.7mM MgSO4 (Fluka; Sleez; Hanover; Germany); 10 mM glucose (Serva); 10 mM Na-HEPES (Serva). Following the removal of connective tissue and adherent fat, the aorta was cut to 1 mm sections, and aorta-rings were digested with collagenase (Collagenase type I., Worthington) for 25 minutes at 37°C. VSMCs were allowed to grow onto 10 cm cell culture plates from the explants for 7-14 days incubated at 5% CO2 and 37°C. VSMCs were cultured in Dulbecco's modified Eagle media (DMEM High Glucose W/ L-Glutamine W/Sodium Pyruvate; Biosera) containing 10% fetal bovine serum (FBS; Biosera), 1% penicillin-streptomycin (Sigma-Aldrich) and 1% GlutaMAX (Gibco). The VSMCs were passaged with trypsin (EuroClone, Milan, Italy) and were used between passages 2-3. Primary rat vascular smooth muscle cells were serum deprived overnight prior to hormone stimulus. Vascular smooth muscle cells (VMSCs) were stimulated with 100 nM Angiotensin II for 2 hours at 37°C. VSMCs were washed twice with cold sterile PBS (137 mM NaCl; 2.7 mM KCl 2.7; 10.1 mM Na2HPO4; 1.8 mM KH2PO4, pH 7.4). Cell lysis was carried out using Trizol reagent (ThermoFisher Scientific). Trizol extraction of total RNA was performed according to the manufacturer's instructions (ThermoFisher Scientific). Poly-A RNAs were captured by oligo-dT conjugated magnetic beads; then, the mRNAs were eluted and fragmented at 94°C. First strand cDNA was generated by random priming reverse transcription, and after the second strand synthesis step, double-stranded cDNA was generated. After repairing ends, A-tailing and adapter ligation steps, adapter ligated fragments were amplified in enrichment PCR and finally sequencing libraries were generated.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-07-05
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-07-05
attributes.tag: External Id
attributes.value: SAMEA110177345
attributes.tag: INSDC center alias
attributes.value: Department of Physiology, Semmelweis University, Budapest, Hungary
attributes.tag: INSDC center name
attributes.value: Department of Physiology, Semmelweis University, Budapest, Hungary
attributes.tag: INSDC first public
attributes.value: 2022-07-05T12:04:00Z
attributes.tag: INSDC last update
attributes.value: 2022-07-05T12:04:00Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11863:DMSO_Vehicle_1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_type
attributes.value: vascular smooth muscle cell
attributes.tag: common name
attributes.value: Norway rat
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: thoracic aorta
attributes.tag: replicate
attributes.value: 1
attributes.tag: sample name
attributes.value: E-MTAB-11863:DMSO_Vehicle_1
attributes.tag: sex
attributes.value: male
attributes.tag: stimulus
attributes.value: vehicle