title: Illumina HiSeq 2000 paired end sequencing; RNA sequencing of pancreatic adenocarcinoma (PDAC) xenograft samples
design: RNA sequencing of pancreatic adenocarcinoma (PDAC) xenograft samples
library_construction_protocol: two tumor sampling methods from patients with PDAC: 1- surgery for patients with tumor size < 2cm, and  2- Biopsy followed by Endoscopic Ultrasound-Guided Fine-Needle Aspiration (EUS-FNA) for patients with tumor size > 2cm. this information is given by the \"Resectable\" variable. For operated patients (with surgery), tumor cells were xenografted directly. however, for biopsy cells, they were grown in culture for 1 or 2 days, before transplanted to the nude mouse. The final xenografted cells will be collected when the tumor reach a size of 1 cm. Finally, the \"Differentiation\", \"Fibrosis\", \"Inflammation\" information are estimated on the collected xenograft tissues. Total RNA was extracted using Manual-miRneasy Mini (Qiagen) Libraries were prepared using Illumina's guidelines for the TruSeq Stranded mRNA LT sample prep kit.  Fragmentation was done using the alternative fragmentation protocol in order to produce RNA-seq libraries that are compatible with sequencing on a paired-end 100 base pair flow cell (PE-100bp FC).  The steps in library prep included o Capture of mRNA on oligo-dT-beads, Concomitant fragmentation and primer annealing, First strand synthesis, Second strand synthesis,  3' enzymatic steps resulting in end repair, 3' adenylation, and adapter ligation and Library amplification (PCR) . The strand specificity of the kit is achieved by the presence of dUTP instead of dTTP during the second strand synthesis step. Since the polymerase used in the downstream library amplification step does not incorporate past dUTP, the incorporation of this nucleotide in second strand synthesis effectively quenches the second strand during amplification.  Libraries were QC'ed with respect to concentration and size profile:  The size profile of the libraries was evaluated by QC of 8 of the samples on an Agilent DNA 1000 or high sensitivity chip. The libraries contain approximately 120 nucleotides of adapter sequence and the remaining size of the library is derived from the input RNA. The library concentration was determined using the KAPA qPCR kit (KK4835) and triplicate reactions using three independent 106–fold dilutions of the libraries.
attributes.tag: Experimental Factor: classical
attributes.value: MultiOmicsClassification