title: 9H_ACAGTG_L007_R1_001.fastq
organism: Homo sapiens
description: Protocols: Polysomal and Free fractions from adipose tissue stem cells cultures treated with cyclopamine, purmorphamine or DMSO for 24 hours were prepared according to (Spangenberg et al., 2013). In brief, cells were treated with 0.1 mg/mL cycloheximide (Sigma-Aldrich) for 10 min at 37oC, removed from the culture flasks with a cell scraper, and resuspended in 0.1 mg/mL cycloheximide in PBS. The suspension was centrifuged (2,000 g for 5 min), and the resulting pellet was washed twice with 0.1 mg/mL cycloheximide in PBS. The cells were lysed by incubation for 10 min on ice with polysome buffer (15mM Tris-HCl pH 7.4, 1% Triton X-100, 15mM MgCl2, 0.3M NaCl, 0.1 mg/mL cycloheximide), and the cell lysate was centrifuged at 12,000 g for 10 min at 4oC. The supernatant was carefully isolated, loaded onto 10% to 50% sucrose gradients, and centrifuged at 39,000 rpm (HIMAC CP80WX HITACHI) for 160 min at 4oC. The sucrose gradient was fractionated with the ISCO gradient fractionation system (ISCO Model 160 gradient former), connected to a UV detector to monitor the absorbance at 275 nm, and the polysome profile was recorded. The free and polysomal RNA fractions were extracted by DirectZOL (Zymo Research). Cells and culture conditions Human Adipose-Derived Stem Cells (ASC) from lipoaspirates were purchased from a commercial source (Lonza®, Walkersville, USA; catalog number PT-5006). ADSC from 3 different donnors were cultured in accordance with datasheet instructions. In brief, cells were cultivated in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco Invitrogen®, Carlsbad, California, USA) supplemented with 10% fetal bovine serum (Gibco Invitrogen®, Carlsbad, California, USA) and 4 mM L-glutamine (Gibco Invitrogen®, Carlsbad, California, USA), without antibiotics. The cells were maintained at 37 C in 5% CO2 atmosphere. Cyclopamine (Sigma Aldrich - C4116) was dissolved in dimethyl sulfoxide, stocked 1mM and used at 5µM in culture for 24 hours. Purmorphamine (Sigma Aldrich - SML0868) was dissolved in dimethyl sulfoxide, stocked 1mM and used at 1µM in culture for 24 hours. The RNA from polysome and free fractions was extracted using DirectZol kit (Zymo). Illumina® TruSeq® Stranded Total RNA Sample Preparation kits
attributes.tag: ENA first public
attributes.value: 2018-08-24
attributes.tag: ENA last update
attributes.value: 2017-11-27
attributes.tag: External Id
attributes.value: SAMEA104406628
attributes.tag: INSDC center alias
attributes.value: FIOCRUZ - PARANA - Instituto Carlos Chagas
attributes.tag: INSDC center name
attributes.value: FIOCRUZ - PARANA - Instituto Carlos Chagas
attributes.tag: INSDC first public
attributes.value: 2018-08-24T17:03:00Z
attributes.tag: INSDC last update
attributes.value: 2017-11-27T15:38:05Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6254:9H_ACAGTG_L007_R1_001.fastq
attributes.tag: age
attributes.value: 38
attributes.tag: batch
attributes.value: Lot 2F3051
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: adipose tissue derived mesenchymal stem cell
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: normal
attributes.tag: organism part
attributes.value: adipose tissue
attributes.tag: sample name
attributes.value: E-MTAB-6254:9H_ACAGTG_L007_R1_001.fastq
attributes.tag: sex
attributes.value: female