title: GSM3590951: neurons_WWOX_del; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted with TRIzol reagent. The libraries for sequencing were prepared according to Piero Carnici's protocol [Takahashi, et al., PMID:22362160]. Briefly, polyadenylated and non-poliadenylated RNA was reverse transcribed into cDNA with usage of random prime containing EcoP15I sequence. Next step was biotinylation of the cap site and the 3'ends of hybridized RNAs. Non-hybridized ssRNAs were digested and 5' complete cDNAs hybridized to biotin-tagged, cap-containing RNAs were separated on streptavidin coated magnetic beads. cDNA was released from RNA and a double stranded 5' linker with a barcode and EcoP15I sequences was ligated. Subsequently, using the biotinylated 2nd SOL primer, a second strand cDNA was synthetized. The digestion with EcoP15I allowed to cleave 27 bp fragments inside the 5' end of the cDNA. In the 3' end of cDNA a 3' linker with 3' Ilumina primer sequence was ligated. Next, constructed CAGE tags were amplified and purified. The prepared libraries were sequenced on Illumina HiSeq System.