Illumina HiSeq 2500 sequencing; Sequencing of DNA captured with a methyl-CpG binding domain (MBD) of primary colorectal cancers, with paired normal tissue, and liver metastases from different patients.
Sequencing of DNA captured with a methyl-CpG binding domain (MBD) of primary colorectal cancers, with paired normal tissue, and liver metastases from different patients.
Tissues were prospectively collected. Biopsies from normal mucosa, colorectal cancers and liver metastases were frozen in liquid nitrogen immediately after surgical resection and stored at -80C. Laser tissue microdissection was performed using the MMI (Molecular Machines & Industries, Glattbrugg, Switzerland) CellCut instrumentation. Briefly, 10 µm-thick sections were cut from frozen tissues embedded in Tissue-Tek O.C.T. (i.e., optimum cutting temperature formulation; Sakura, Alphen aan den Rijn, The Netherlands) using the Hyrax C60 cryostat (Zeiss, Feldbach, Switzerland) and placed on MMI membrane slides. Sections on these slides were fixed in propanol for 45 seconds, covered with one drop of Mayer's hematoxylin (MHS128, Sigma-Aldrich, Buchs, Switzerland) for 45 seconds, vigorously washed, sequentially dipped in 0.1% ammonia (10 seconds), propanol (45 seconds) and xylene (45 seconds), and finally dried for 5 minutes. This staining was immediately followed by laser tissue microdissection using special MMI tubes with caps on which dissected sections adhere. Only epithelial normal crypts or tumor glands were selected, cut and collected on the cap, minimizing thus contamination with stromal cells. Immediately after dissection, DNA extraction was performed with QIAmp DNA Micro kit (Qiagen, Hombrechtikon, Switzerland) and the extracted DNA quantified with Qubit fluorometer and dsDNA HS Assay kit (Thermo Fisher Scientific, Reinach, Switzerland). Methylated DNA was captured and sequenced using MBD-isolated (methyl-CpG binding domain) genome sequencing with scaling down reaction volumes to successfully process the small amount of genomic DNA extracted with laser tissue microdissection. Briefly, 100 ng of input DNA was fragmented to an average length of 200 bp the Covaris S2 ultrasonicator. Enrichment of methylated DNA was performed with the MethylMiner Kit (Invitrogen, city, country) using a single elution step with 2000 mM NaCl elution buffer. The NEBNext Ultra DNA Library Prep Kit was used for multiplex Illumina library production. The size (average length 350 bp) and the concentration of libraries were evaluated with the Agilent 2200 TapeStation system.