GSM7124305: T_cells_ChronicallyActivated_stimulated_Donor2; Homo sapiens; RNA-Seq
Frozen cell pellets were re-suspended in lysis buffer from the GenElute™Mammalian Total RNA Miniprep Kit (SIGMA). mRNA was purified following manufacturer's instructions. The RNA sample was checked for quality and quantity using a NanoDrop spectrophotometer and an Agilent Bioanalyzer. Poly(A) mRNA was enriched from the total RNA using oligo(dT) magnetic beads. The enriched mRNA was fragmented into smaller pieces using divalent cations under elevated temperature. The fragmented mRNA was used as a template to synthesize first-strand cDNA using random hexamer primers, followed by second-strand cDNA synthesis using DNA polymerase I and RNase H. he double-stranded cDNA was treated with T4 DNA polymerase and Klenow DNA polymerase to create blunt ends, followed by adapter ligation using T4 DNA ligase. The adapter-ligated cDNA fragments were size-selected using agarose gel electrophoresis or bead-based methods, and then PCR amplified to generate the final cDNA library.