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: Small Intestine_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).  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: small intestine
attributes.tag: Experimental Factor: disease
attributes.value: normal
attributes.tag: Experimental Factor: cell line
attributes.value: not applicable
attributes.tag: Experimental Factor: stimulus
attributes.value: none