GSM3611501: N7 [RNA-Seq]; Homo sapiens; RNA-Seq
RNA extraction was performed using the miRNeasy Isolation Kit (Quiagen) for miRNA and Total RNA isolation, according to the manufacturer's instructions from human tumor and non-transformed tissue. 500 ng of total RNA was depleted of ribosomal RNA using the RiboMinus kit (Life Technologies) according to the instructions of the manufacturer. Depleted RNA was then fragmented by addition of 5  fragmentation buffer (200 mM Tris acetate, pH 8.2, 500 mM potassium acetate and 150 mM magnesium acetate) and heating at 94 °C for 3 min in a thermocycler followed by ethanol precipitation with ammonium acetate and GlycoBlue (Life Technologies) as carrier. Fragmented RNA was then reversed transcribed using random hexamer and Superscript III (Life Technologies). The second strand was synthesized using the TargetAmp kit (Epicentre) according to the instructions of the manufacturer. The final steps of library preparation, e.g. blunt end repair, adapter ligation, adapter fill-in and amplification were done according to Meyer and Kircher (2010, PMID: 20516186). The barcoded libraries were purified and quantified using the Library Quantification Kit - Illumina/Universal (KAPA Biosystems) according to the instructions of the manufacturer. A pool of up to 10 libraries was used for cluster generation at a concentration of 10nM using an Illumina cBot. Sequencing of 2x100 bp was performed with an IlluminaHighScan-SQ sequencer at the sequencing core facility of the IZKF Leipzig (Faculty of Medicine, University Leipzig) using version 3 chemistry and flowcell according to the instructions of the manufacturer.
GEO Accession
GSM3611501