GSM4815225: Nt Hypoxia rep1; Homo sapiens; RNA-Seq
Total RNA was extracted with nucleozol reagent Total RNA samples were prepared and their concentration was measured in nanodrop (ND1000 Spectrophotometer – PEQLAB). The concentration required for library preparation varies from 5 to 500ng per 5μl. Samples with a concentration of more than 500ng/μl, were diluted accordingly to a mean concentration of approximately 100-150ng/μl. The RNA quality of each sample was measured in bioanalyzer (Agilent Technologies) using the Agilent RNA 6000 Nano Kit reagents and protocol. (RNA samples with RNA Integrity Number (RIN) >7 are preferred, although RNAs of lower quality can also be used, and further processed for library preparation and sequencing).  For library preparation, the 3' mRNA-Seq Library Prep Kit Protocol and reagents, for Ion Torrent (QuantSeq-LEXOGEN™ Vienna, Austria) was used according to manufacturer's instruction. Briefly, library generation is initiated by oligodT priming which contains the Ion Torrent compatible linker sequences. Approximately 300ng per 5μl of RNA from each sample was used to perform the first strand synthesis. Any remaining RNA is removed and 2nd strand synthesis is initiated by a random primer, containing Ion Torrent compatible linker sequences at its 5' end, and a sequence polymerase. In line barcodes are introduced at this point. 2nd strand synthesis is followed by a magnetic bead-based purification step and the resulted purified library is amplified for 13 to 18 cycles and re-purified.  Each library's quality and quantity was assessed in bioanalyzer using the DNA High Sensitivity Kit reagents and protocol (Agilent Technologies). The quantified libraries were pooled together in 12plex at a final concentration of 7pM.  The libraries pool was processed on the Ion Proton One Touch system where the libraries were templated and enriched with the Ion PI™ Hi-Q™ OT2 200 Kit (ThermoFisher Scientific) and sequenced, with the Ion PI™ Hi-Q™ Sequencing 200 Kit on Ion Proton PI™ V2 chips (ThermoFisher Scientific) according to commercially available protocols. 3'RNA-sequencing is performed on an Ion Proton™ System (Rothberg et al, 2011), according to the manufacturer's instructions.   Initial analysis took place in Ion Torrent server where the strand specific sequencing reads mapped to their corresponding strand on the genome. 3'RNA-sequencing
GEO
GSM4815225