alias: A29399-1
title: RNA-Seq analysis of brain glioblastoma multiforme sample GBM_62 (A29399)
design: RNA-Seq analysis of brain glioblastoma multiforme sample GBM_62 (A29399) using Illumina HiSeq 2000
library_construction_protocol: PolyA+ mRNA was column purified (including on column DNaseI treatment) using the MACS 96 Separation Unit (Miltenyi Biotec) as per the manufacturer's protocol. 2-10 ug of total RNA with a RIN>=7 (Agilent Bioanalyzer) were used as input. The purified polyA+ RNA was used as substrate for double-stranded cDNA synthesis using the Superscript II Double-Stranded cDNA Synthesis kit (Life Technologies) and 200ng random hexamers (Life Technologies). Double stranded cDNA was purified using 2 volumes of Ampure XP beads, fragmented using Covaris E series shearing (20% duty cycle, Intensity 5, 55 seconds), and used for paired-end sequencing library preparation (Illumina). Prior to library amplification uridine digestion was performed at 37C for 30 min following with 10 min at 95C in Qiagen Elution buffer (10mM Tris-Cl, pH 8.5) with 5 units of Uracil-N-Glycosylase (UNG: AmpErase). The resulting single stranded sequencing library was amplified by PCR (10-13 cycles) to add Illumina P5 and P7 sequences for cluster generation. PCR products were purified on Qiaquick MinElute columns (Qiagen) and assessed and quantified using an Agilent DNA 1000 series II assay and Qubit fluorometer (Life Technologies) respectively.
identifiers.namespace: BCCAGSC
identifiers.id: A29399-1