GSM8808023: patient 417, tumor tissue, former smoker; Homo sapiens; RNA-Seq
Total RNAs were extracted from 5mm lung tissue or tumor using Trizol (phenol-chloroform extraction, Qiagen kit). Extracted RNAs were resuspended in 50ul of DEPC-treated water and stored in -80 C until library preparation mRNAs were purified from approximately 200ng of total RNA with oligo-dT beads and sheared by incubation at 94C in the presence of Mg. Following first-strand synthesis with random primers, second-strand synthesis and A-tailing were performed with dUTP for generating strand-specific sequencing libraries. Adapter ligation with 3' dTMP overhangs was ligated to library insert fragments.  Library amplification amplified fragments carrying the appropriate adapter sequences at both ends.  Strands marked with dUTP were not amplified. Indexed libraries were quantified by qRT-PCR using a commercially available kit (Roch KAPA Biosystems Cat# KK4854) and insert size distribution was determined by either the Agilent Bioanalyzer. Samples with a yield of ≥ 0.5 ng/ul and a size distribution of 150-300bp were used for sequencing.