ST4_P6
Homo sapiens
Protocols: 50 ml of urine was collected from 15 patients of various stages of chronic kidney disease and 10 healthy controls.To remove the urinary cell debris or cells, samples were centrifuged at 1000 rpm for 10 min, followed by 2500 rpm for 10 min at 4°C. The supernatants were transferred to Beckmann tubes and centrifuged at 187 000 x g for 1 hr and 30 min at room temperature. The supernatants were saved and pellets were resuspended in 1 ml isolation solution [250 mM sucrose, 10 mM triethanolamine (pH 7.6)], followed by incubation with 50 µl 1 M dithiothreitol (DTT). Samples were vortexed vigorously, resuspended in supernatant and centrifuged again at 187 000 x g for 1 hr and 30 min at room temperature. Exosomes were resuspended in 100 µl DEPC-treated water and used for total RNA extraction. DEPC-treated water was added to samples to a final volume of 400 μl. Equal amounts of Tri-reagent (Sigma, USA) were added to each sample, and the mixture was incubated at 95oC for 5 min, cooled on ice to 4oC for 5 min, and incubated at room temperature for another 5 min. One volume of phenol chloroform isoamyl alcohol solution was added to the samples, which were centrifuged for 5 min at 13 000 rpm. The aqueous phase was carefully removed and transferred to a fresh tube. To remove residual phenol, a second chloroform extraction was carried out. Subsequently, the RNA was precipitated in two and a half volumes of ethanol and one-tenth volume of sodium acetate (3 M, pH 5.2). Samples were incubated at -80oC for 20 min and centrifuged at 13 000 rpm, 4oC, for 20 min. Pellets were washed with 70% ethanol, centrifuged at 13 000 rpm for 5 min and dissolved in 20 µl DEPC-treated water. For each sample, 10 ng of total RNA was reverse transcribed into cDNA, as described in the manufacturer's instruction. The libraries were generated with Ion Total RNA-Seq Kit v2 following the manufacturer's instructions, inhibiting cDNA synthesis of the adaptor byproducts and cDNA separation with magnetic bead-based technology. The samples are pooled and loaded on the Ion chip Kit v2 and sequenced on the Ion Proton sequencer
ENA first public
2016-11-30
ENA last update
2016-10-12
External Id
SAMEA4504177
INSDC center alias
Innsbruck Medical University
INSDC center name
Innsbruck Medical University
INSDC first public
2016-11-30T17:01:27Z
INSDC last update
2016-10-12T10:47:27Z
INSDC status
public
Submitter Id
E-MTAB-5152:ST4_P6
age
61
broker name
ArrayExpress
common name
human
disease
chronic kidney disease
disease staging
Stage IV
organism part
urine
sample name
E-MTAB-5152:ST4_P6
sex
male