RRBS Protocol for Genome-Wide DNA Methylation

RRBS was performed as described previously51 (link),52 , starting with 100 ng of genomic DNA per sample. Custom-designed methylated and unmethylated oligonucleotides were added at a concentration of 0.1% to serve as spike-in controls for monitoring bisulfite conversion efficiency. After adaptor ligation, RRBS libraries were quantified by qPCR and pooled in combinations of six. For library enrichment, the number of PCR cycles was determined by qPCR and never exceeded 18 cycles. The library was purified twice using Agencourt AMPure XP beads (Beckman Coulter, A63880). Quality control for the final library was performed by measuring the DNA concentration with the Qubit dsDNA HS assay (ThermoFisher Scientific, Q32851) on Qubit 2.0 Fluorometer (ThermoFisher Scientific, Q32866) and by determining library fragment sizes with the Experion DNA 1K Analysis kit (Bio-Rad, 700-7107) on the Experion Automated Electrophoresis Station (Bio-Rad, 701-7000). Libraries were sequenced on Illumina HiSeq 2000/2500 machines.

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Sheffield N.C., Pierron G., Klughammer J., Datlinger P., Schönegger A., Schuster M., Hadler J., Surdez D., Guillemot D., Lapouble E., Freneaux P., Champigneulle J., Bouvier R., Walder D., Ambros I.M., Hutter C., Sorz E., Amaral A.T., de Álava E., Schallmoser K., Strunk D., Rinner B., Liegl-Atzwanger B., Huppertz B., Leithner A., de Pinieux G., Terrier P., Laurence V., Michon J., Ladenstein R., Holter W., Windhager R., Dirksen U., Ambros P.F., Delattre O., Kovar H., Bock C, & Tomazou E.M. (2017). DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma. Nature medicine, 23(3), 386-395.

Publication 2017

AMPure XP beads Qubit dsDNA HS assay Qubit 2.0 Fluorometer Experion DNA 1K Analysis kit Experion Automated Electrophoresis Station HiSeq 2000/2500 machines

Assay Bisulfite Dsdna Electrophoresis Genomic Library Ligation Oligonucleotides

Corresponding Organization : Medical University of Vienna

Other organizations : CeMM Research Center for Molecular Medicine, Austrian Academy of Sciences, Université Paris Sciences et Lettres, Institut Curie, Inserm, Centre Hospitalier Régional et Universitaire de Nancy, St Anna Children's Hospital, Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío, Paracelsus Medical University, Medical University of Graz, Centre Hospitalier Universitaire de Tours, Institut Gustave Roussy, Vienna General Hospital, University Hospital Münster

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Variable analysis

independent variables

Amount of genomic DNA per sample (100 ng)

dependent variables

Bisulfite conversion efficiency
DNA concentration of final library
Library fragment sizes

control variables

Concentration of custom-designed methylated and unmethylated oligonucleotides (0.1%)

controls

Positive control: Custom-designed methylated oligonucleotides
Negative control: Custom-designed unmethylated oligonucleotides

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