title: GSM4783610: MD17227_A; Homo sapiens; RNA-Seq
library_construction_protocol: After sampling normal mucosa samples were immediately placed in RNAlater and kept immersed for 24-72 hours prior to RNA extraction. RNA was then extracted from NM and PBMC samples using a TRIzol based proprietary RNA extraction kit (Ribopure kit, Applied Biosystems) according to manufacturer protocol. 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. The fragmentation time was shorten from 8 minutes to 1 minutes to allow for bigger RNA fragments. 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.