Sample 17
Protocols: Cells were transfected at 80% confluence using a mixture of siLenfect (Bio-Rad Laboratories AB, Solna, Sweden) and siRNA (20 nM), according to the manufacturer (Dharmacon/GE Healthcare, Uppsala, Sweden). Forty-eight hours after transfection cells were either stimulated with 5ng/ml TGFβ or 30ng/ml BMP7, or were unstimulated as control. The patient-derived glioblastoma cells U3034MG were cultured in adherent conditions on laminin (Sigma-Aldrich Sweden AB, Stockholm, Sweden), in DMEM/F12 Glutamax (GIBCO, Life Technologies Europe BV, Stockholm, Sweden) and Neurobasal medium (GIBCO, Life Technologies Europe BV, Stockholm, Sweden) mixed in 1:1 ratio, with the addition of 1% B27 (Invitrogen, Life Technologies Europe BV), 1% N2 (Invitrogen, Life Technologies Europe BV), 1% penicillin/streptomycin (Sigma-Aldrich Sweden AB, Stockholm, Sweden), 10 ng/ml EGF and FGF2 (PeproTech, EC Ltd, London, UK). Cellular RNA from ~250,000 cells was purified with the Nucleospin RNA kit ((Macherey-Nagel, Düren, Germany). RNA concentration was measured with ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Total RNA for Ampliseq was extracted from 3 biological replicates as described above. Fifty ng of RNA was reverse transcribed to cDNA using the Ion AmpliSeq Transcriptome Human Gene Expression Kit Preparation protocol (Revision A.0, Life Technologies). The acquired cDNA was amplified using Ion AmpliSeq Transcriptome Human Gene Expression core panel (Life Technologies) and the primer sequences were then partially digested. Then, DNA adaptors (Ion P1 Adaptor and Ion Xpress Barcode Adaptor, Life Technologies) were ligated to the amplicons. Adaptor ligated amplicons were purified using the Agencourt AMPure XP reagent (Beckman Coulter) and eluted in amplification mix (Platinum PCR SuperMix High Fidelity and Library Amplification Primer Mix, Life Technologies) and amplified. Size-selection and purification was conducted using Agencourt AMPure XP reagent (Beckman Coulter).
ENA first public
2021-05-12
ENA last update
2021-05-12
External Id
SAMEA7625093
INSDC center name
UU
INSDC first public
2021-05-01T00:23:53Z
INSDC last update
2020-11-25T14:57:04Z
INSDC status
public
Submitter Id
E-MTAB-9813:Sample 17
broker name
ArrayExpress
cell line
U3034MG
common name
human
developmental stage
adult
disease
brain glioblastoma
genotype
wild type genotype
organism part
brain
replicate
3
sample name
E-MTAB-9813:Sample 17