title: Illumina HiSeq 2500 sequencing; Expression profiling of budding cells in colorectal cancer suggests an EMT-like phenotype and molecular subtype switching
design: Expression profiling of budding cells in colorectal cancer suggests an EMT-like phenotype and molecular subtype switching
library_construction_protocol: Eight cases of colorectal cancer were selected from the archive of the pathology department of the University Hospitals of Leuven (Belgium). Inclusion criteria were: (1) diagnosis of colorectal cancer was established according to the criteria of the World Health Organisation (WHO) (2) diagnosis was made between 2003 and 2012 (3) patients were free from pre-operative radio or chemotherapy to avoid interference with the grade of tumour budding and gene expression profile (4) availability of FFPE material (5) availability of matched fresh frozen (FF) tissue (6) high tumour budding was observed in both FFPE and FF tissue at the invasive margin of the tumour (7) good or moderate (non-mucinous) differentiation pattern. RNA sequencing was performed on 8 budding and 8 matched main tumors using the TruSeq RNA access Library Prep Kit (Illumina) following to the manufacturers instructions. In short, RNA is fragmented using divalent cations at 94oC for 8 minutes. The cleaved RNA fragments are then converted to cDNA using random priming during first and second strand synthesis. An A-base is then added to the resulting double-stranded cDNA fragments and sequencing adapters are ligated. The resulting library is amplified using PCR. Subsequently, 2 rounds of 90 minutes hybridization at 58oC with sequence-specific probes were used to capture the coding regions of the transcriptome and bind them to streptavidin magnetic beads. Two heated washes remove non-specificly bound fragments from the beads after which the captured fragments are again enriched with PCR.
attributes.tag: Experimental Factor: bud cell
attributes.value: developmental stage