GSM1753021: Alzheimer's Brain Sample63; Homo sapiens; RNA-Seq
RNA was extracted with the SPLIT RNA extraction kit (Lexogen GmbH, Vienna, Austria) yielding the large RNA fraction with a lower cut-off size of 150 nt. Evaluation on a Bioanalyzer RNA Nano chip (Agilent Technologies) showed medium to high RNA quality (RIN of 6.2 – 8.3). 4 µg RNA was incubated with Terminator 5´-phosphate-dependent exonuclease (Epicentre, Madison, WI, USA) to remove degraded RNA while leaving the capped, full-length mRNA intact Per sample, 20 ng of the purified full-length total RNA were used for performing the 3’ QuantSeq Reverse protocol (Lexogen GmbH, Vienna, Austria) resulting in NGS libraries which originate from the 3’ end of polyadenylated RNA. Library generation was started by oligo(dT) priming with primers already containing the Illumina-compatible linker sequence for Read 1. After first strand synthesis the RNA was removed before the second strand synthesis was initiated by random primers which contained the corresponding Illumina-compatible linker sequence. 3’ QuantSeq generated just one fragment per transcript at the very 3’ end. The libraries were PCR amplified and barcoded in 18 cycles. Equal amounts of the 72 libraries were combined to one lane mixture.