Homo sapiens
Protocols: Normal human tissue RNA was obtained from the FirstChoice Tissue Panel (Ambion AM6000) and Human Breast Total RNA (Ambion AM6952). Melanoma RNA was obtained from patient cell lines, including 3 originating from metastasized stage IV melanoma (A-series) and 10 originating from resected lymph nodes from patients with stage 3 disease (C-series. RNA was extracted with RNeasy columns (Qiagen). Breast cancer cell lines were grown in RPMI medium supplemented with 10% fetal calf serum (FCS), sodium pyruvate, 10g/ml insulin and antibiotics. Normal breast epithelial cell line, Bre-80 (provided as a gift from Roger Reddel, CMRI, Sydney) was grown in DMEM/F12 medium with 5% horse serum (HS), 10g/ml insulin, 0.5g/ml hydrocortisone, 20ng/ml epidermal growth factor, 100ng/ml cholera toxin and antibiotics. Cell lines were maintained under standard conditions routinely tested for Mycoplasma and short tandem repeat (STR) profiled for authentication. RNA was extracted with RNeasy columns (Qiagen). RNA was DNAse treated with TurboDNase (Life Technologies), confirmed DNA-free, RNA integrity was confirmed by Agilent 2100 Bioanalyzer (Agilent Technologies). rRNA depletion (Ribo-Zero™ (Epicentre)) was performed on 5μg of total RNA. ERCC RNA Spike-In Control mix 1 or mix 2 (Life Technologies) were added to ribodepleted RNA to a final dilution of 1/100. Libraries were prepared with the TruSeq Stranded mRNA Sample Preparation Kit (Illumina) and 9-12 cycles of pre-capture LM-PCR performed as required. Multiplex library pools were created by mixing equal amounts of 5 pre-capture sample libraries and capture hybridization performed on 1 μg of the pooled library. Melanoma A-series samples were each randomly assigned to one of three capture hybridizations to ensure any expression differences between the A and C-series samples were not due to an A-series batch effect. Post-capture LMPCR was performed for 17 cycles.
ENA first public
2017-07-04
ENA last update
2017-07-03
External Id
SAMEA104150451
INSDC center alias
Garvan Institute of Medical Research
INSDC center name
Garvan Institute of Medical Research
INSDC first public
2017-07-04T17:02:27Z
INSDC last update
2017-07-03T09:35:25Z
INSDC status
public
Submitter Id
E-MTAB-4519:BT474
broker name
ArrayExpress
cell line
BT474
common name
human
disease
breast carcinoma
organism part
breast
sample name
E-MTAB-4519:BT474