title: Illumina HiSeq 2000 paired end sequencing; Intergenic Disease-associated Regions are Abundant in Novel Regulatory Transcripts
design: Intergenic Disease-associated Regions are Abundant in Novel Regulatory Transcripts
library_name: T47D_p
library_construction_protocol: 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.
attributes.tag: Experimental Factor: organism part
attributes.value: breast
attributes.tag: Experimental Factor: disease
attributes.value: breast carcinoma
attributes.tag: Experimental Factor: cell line
attributes.value: T47D
attributes.tag: Experimental Factor: stimulus
attributes.value: none