GSM1575302: DGM-01355_AM; Homo sapiens; RNA-Seq
Total RNA from harvested nonsmoker, smoker and COPD alveolar macrophages was extracted with TRIzol with subsequent RNA clean-up using the RNeasy MinElute RNA purification kit to remove residual DNA Using the reagents of the mRNA Sample Prep Kit and in accordance with the RNA sequencing protocol provided by Illumina, poly(A)+ mRNA was selected out from the total RNA samples using Sera-mag magnetic oligo(dT) beads. An RNA fragmentation kit (Ambion, Austin, TX) was used to fragment the mRNA, followed by first- and second-strand cDNA synthesis using random hexamer primers. An “end repair” reaction to blunt the ends of all fragments was then performed with Klenow polymerase and T4 DNA polymerase, and 3’- to 5’ exo-nuclease was used to create a 3’ adenine overhanging tail, facilitating the ligation of the amplification adapters. Ligation products were then separated on a 2% tris-acetate-EDTA-agarose gel for size selection, followed by purification with a gel extraction kit. The purified ligation products were then PCR amplified with complementary primers and the resultant cDNA was purified with QIAquick PCR kit (Qiagen), and the concentration was measured by the NanoDrop spectrophotometer. Samples were then loaded onto Illumina flow cells for single end, 43 nucleotide, sequencing reactions Images acquired by the Illumina Genome Analyzer II were analyzed by Firecrest and bases called by Bustard (both part of Illumina RTA pipeline version 1.6).
GEO
GSM1575302