Illumina Genome Analyzer IIx sequencing; Pancreatic ductal adenocarcinoma primary cultures enriched or not in cancer stem cells
Pancreatic ductal adenocarcinoma primary cultures enriched or not in cancer stem cells
PDAC spheres were seeded in complete media, in ultra-low attachment plates (Corning B.V., Schiphol-Rijk, The Netherlands) as described previously (Gallmeier et al., 2011). '2 Âľg of total RNA samples was used as provided by the user. RNA Integrity Numbers were in the range 9.5 to 10 when assayed on an Agilent 2100 Bioanalyzer. PolyA+ RNA fraction was extracted and randomly fragmented, converted to double stranded cDNA and processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters as in Illumina''s \"TruSeq RNA Sample Preparation v2 Protocol\" (Part # 15026494 Rev. C). Adapter-ligated library was completed by 8 cycles of PCR with Illumina PE primers. The resulting purified cDNA library was applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and sequenced on the Genome Analyzer IIx with SBS TruSeq v5 reagents by following manufacturer''s protocols.'
Experimental Factor: spheroid culture
growth condition
Experimental Factor: cancer stem cell-enriched
phenotype
Experimental Factor: patient 3
individual