Illumina HiSeq 2000 sequencing; Small RNA-Sequencing of PDAC tissues and control samples.
Small RNA-Sequencing of PDAC tissues and control samples.
Tissue samples were obtained from six patients with PDAC who underwent resection at the Department of Surgery, Technical University of Munich, Germany.  Normal pancreatic tissue samples from five patients without pancreatic ductal adenocarcinoma were used as controls.   Tissue collection was approved by the Ethics Committee of the Technical University of Munich and informed consent was obtained from all patients. Tissue were collected directly in the operating theatre and were immediately stored at -80 degree C until analysis. 20 mg of frozen tissue were disrupted and homogenized (TissueLyser, Qiagen) and RNA was isolated (NucleoSpin miRNA Kit, Macherey-Nagel) in two fractions (small RNA < 200 nt and large RNA > 200 nt). For preparation of small RNA libraries, 5 ug RNA (small RNA fraction) was size-selected (< 40 nt) by polyacrylamide gel electrophoresis (FlashPAGE, Life Technologies) and precipitated. About 30 ng small RNA (< 40 nt) was successive ligated (T4 RNA Ligase 1 and T4 RNA Ligase 2, NEB) to modified 3 prime and 5 prime adapters (TrueQuant RNA adapters, GenXPro). Adapter-ligated RNA was reverse transcribed (SuperScript III, Life Technologies) and amplified by PCR (KAPA HiFi Hot-Start Polymerase, KAPA Biosystems). Amplified libraries were size-selected by polyacrylamide gel electrophoresis (PAGE) .
Experimental Factor: disease
pancreatic ductal adenocarcinoma
LIBRARY_STRAND
first strand