title: RNA-Seq of mRNA contents of IDC patient 2
design: Plasma samples from breast cancer patients and healthy donors were obtained through an Institutional Review Board approved protocol in compliance with the Declaration of Helsinki.  All subjects provided written informed consent for blood to be used for research purposes. EVs were isolated from 1 ml of plasma per patient by differential centrifugation. The 10,000 g and 100,000 g pellets were collected in TRIzol reagent (Invitrogen) and combined for RNA isolation.  Approximately 1 to 10 ng of total RNA from the EV samples was used for library preparation.  These libraries were made using a pre-release version of the Illumina RNA Access kit (Ref = http://www.illumina.com/products/truseq-rna-access-kit.html).  Briefly, this protocol uses random priming to create 1st and 2nd strand cDNA from total RNA.  No poly-A selection or rRNA depletion is done prior to creation of cDNA. The total cDNA libraries are then enriched for protein coding regions of mRNA by hybridization and capture using a set of probes designed against 21, 415 human genes.