title: Illumina HiSeq 2500 paired end sequencing; The epigenetic landscape of human colorectal cancer
design: The epigenetic landscape of human colorectal cancer
library_construction_protocol: Primary colonic normal and tumoral tissues were obtained from the hospital. Normal and tumoral tissues were processed according to Fujii et al (Fujii et al, Nature Protocol 2015). Briefly, surgically resected specimens were reduced to 3-5 mm size, extensively washed with cold PBS and gentamicin (20g/ml) and incubated with PBS-EDTA rocking for 1h at 4°C. After PBS-EDTA, tissues samples were further washed with cold PBS and normal crypts and tumoral cells were released and collected by centrifugation at 400g for 5 minutes 4°C. For RNA-seq analysis, cells suspension from CRC PDOs, primary normal and tumoral tissues were lysed in TRIzol reagent (Thermo Fisher) and processed for total RNA extraction with PureLink™ RNA Mini Kit (Thermo Fisher), according to manufacturer's procedure. PDOs samples were collected at early (<5 splits) and late passages (>5 splits). The RNA quality was assessed by the RNA Integrity Number (RIN) value with RNA6000 assay (Agilent). Only samples with RIN>7.0 were used in this study.  Libraries were prepared using the TruSeq mRNA Stranded preparation kit (Illumina, San Diego, USA).