GSM4217438: MKG109_OVCAR8 AZ_300:3; Homo sapiens; RNA-Seq
Cells were grown in 25cm2 flasks until they reached 70% confluency then total RNA was extracted using the RNeasy mini kit (74104, Qiagen Ltd., Manchester, UK) according to the manufacturers' instructions. Libraries were prepared from each total-RNA sample using the TruSeq Stranded Total RNA with Ribo-Zero Gold kit (#RS-122-2302) according to the provided protocol.  500ng of total-RNA was processed to deplete rRNA before being purified, fragmented and primed with random hexamers. Primed RNA fragments were reverse transcribed into first strand cDNA using reverse transcriptase and random primers. RNA templates were removed and a replacement strand synthesised incorporating dUTP in place of dTTP to generate ds cDNA. AMPure XP beads (Beckman Coulter, #A63881) were then used to separate the ds cDNA from the second strand reaction mix, providing blunt-ended cDNA. A single 'A' nucleotide was added to the 3' ends of the blunt fragments to prevent them from ligating to another during the subsequent adapter ligation reaction, and a corresponding single 'T' nucleotide on the 3' end of the adapter provided a complementary overhang for ligating the adapter to the fragment. Multiple indexing adapters were then ligated to the ends of the ds cDNA to prepare them for hybridisation onto a flow cell, before 12 cycles of PCR were used to selectively enrich those DNA fragments that had adapter molecules on both ends and amplify the amount of DNA in the library suitable for sequencing.
GEO Accession
GSM4217438