title: GSM2857276: Lung_tumor [SVA501]; Homo sapiens; RNA-Seq
library_construction_protocol: Frozen lung tissue samples ranging in weight from 25-60 mg were individually ground to a fine powder using a liquid nitrogen cooled ceramic mortar and pestle. For RNA quality control (QC), approximately 20% of each sample was processed with TRI Reagent (Sigma Aldrich, St Louis, MO). Once the initial RNA QC was completed, polyribosome preparations were performed in random sample pairs. For this purpose, the remainder of each frozen powdered sample was solubilized in 100 µL of lysis buffer (10 mM Tris-HCl at pH 8.0, 150 mM NaCl, 5 mM MgCl2, 1% Nonidet-P40, 1% sodium deoxycholate, 40 mM dithiothreitol, 500 U/mL RNAsin (Promega, Madison, WI), 40 mM VRC (vanadyl ribonucleoside complex), and 150 µg/ml cycloheximide) and mixed by pipetting. Nuclei and insoluble material were removed by centrifugation (12,000 x g, 10 seconds, at 4°C). Extraction buffer (50 µL; 0.2 M Tris-HCl at pH 7.5, 0.3 M NaCl, 10 mM PMSF) was added to the supernatant and the sample was mixed by pipetting. The sample was centrifuged (12,000 x g, 5 min, at 4° C) and 25% of the supernatant was processed with TRI Reagent to generate a cytosolic total RNA sample. The remaining supernatant was layered onto a 5 mL, linear sucrose gradient (0.5–1.5 M), 10 mM Tris-HCl pH 7.5, 140 mM NaCl, 3 mM MgCl2, 10 mM DTT. Gradients were centrifuged (200,000 x g in a Beckman SW55Ti rotor for 80 minutes at 4o C) and the gradients were fractionated into ten, 0.5 mL fractions utilizing an ISCO density gradient fractionator with absorbance monitored at 254 nm. Each fraction was collected into a tube containing 50 µL 1%SDS and 20 µL 0.5 M EDTA. The polyribosomal mRNA from fractions 7, 8, 9 and 10 were individually processed with 1 mL of TRI Reagent and 200 µL chloroform and the resultant RNA pellets from these four fractions were combined to create the polyribosomal mRNA sample corresponding to each tissue sample. Polyribosomal mRNA and cytosolic RNA were sent to the University of Minnesota Genomics Core for quality assessment by Caliper High Sensitivity Lab Chip. Those samples passing the Caliper QC were processed into indexed library samples for analysis by RNA sequencing. This was accomplished using the SMART-Seq protocol. Libraries were quantified using a fluorimetric PicoGreen assay. Library size was assessed using capillary electrophoresis on an Agilent BioAnalyzer 2100 and only libraries quantifying higher than 10 nM were analyzed. Libraries were pooled and assessed using Kapa qPCR to ensure adapters were ligated to DNA fragments and functional. Nextera libraries were hybridized to a single read flow cell and individual fragments were clonally amplified by bridge amplification on the Illumina cBot. Once clustering was complete, the flow cell was loaded on a HiSeq 2000 and sequenced using Illumina SBS chemistry. Upon completion of read 1, an 8-base pair index read for Index 1 was performed. The Index 1 product was removed and the template was reannealed to the flow cell surface. The run proceeded with 8 chemistry-only cycles, followed by an 8-base pair index read to read Index 2.
attributes.tag: GEO Accession
attributes.value: GSM2857276