title: Margin BS-seq part 1
organism: Homo sapiens
description: Protocols: Brain tumour biopsies were fixed in 10% formalin and embedded in paraffin wax from which 4-µm-thick sections were cut and stained with haematoxylin and eosin. The diagnosis of glioblastoma multiforme (WHO grade IV) was made by light microscopical examination of H&E-stained sections. Supra-total resection of a right frontal intrinsic tumour with maximum diameter of the enhancing component of 19mm, surrounded by only minimal oedema. Surgical samples were taken from the enhancing tumour mass and corresponding non-enhancing margin using advanced surgical techniques. PCR-free ReBuilT libraries for bisulfite sequencing were generated following our previously published method (pubmed id: 27031619). For the generation of PCR-free libraries for oxBS, libraries were oxidised (TrueMethyl Kit from CEGX) prior to bisulfite treatment. In brief, 350 and 175 ng of sonicated DNA from tumour and margin respectively were end repaired and dA-tailed before ligation of customized adapter pair. After (oxidative)-bisulfite treatment, degraded fragments were recovered through primer extension before dA-tailing and second adapter ligation. The high-resolution aim of this project led to divide each sample in 3 parts in order to be able to perform sequencing. Parts were subsequently immobilized on streptavidin coated magnetic beads, washed with binding buffer, eluted with 50 mM NaOH at 60°C for 15 minutes. Each tumour and margin sample was frozen to -80°C immediately on collection. The frozen samples were then defrosted and a 50 mg portion taken for DNA/RNA extraction. The samples were homogenised and then divided for DNA and RNA extraction. DNA extraction of both tissues and blood was performed using the QIAGEN DNeasy Blood and Tissue Kit, RNA extraction was performed using the QIAGEN RNeasy Mini Kit. Quantification was performed using the Qubit quantification assay. The tumour/normal pair used in this study was the sample judged most pure by comparison with blood DNA using whole genome sequencing. PCR-free ReBuilT libraries for bisulfite sequencing were generated following our previously published method (pubmed id: 27031619). For the generation of PCR-free libraries for oxBS, libraries were oxidised (TrueMethyl Kit from CEGX) prior to bisulfite treatment. In brief, 350 and 175 ng of sonicated DNA from tumour and margin respectively were end repaired and dA-tailed before ligation of customized adapter pair. After (oxidative)-bisulfite treatment, degraded fragments were recovered through primer extension before dA-tailing and second adapter ligation. The high-resolution aim of this project led to divide each sample in 3 parts in order to be able to perform sequencing. Parts were subsequently immobilized on streptavidin coated magnetic beads, washed with binding buffer, eluted with 50 mM NaOH at 60°C for 15 minutes.
attributes.tag: ENA first public
attributes.value: 2017-02-24
attributes.tag: ENA last update
attributes.value: 2016-10-17
attributes.tag: External Id
attributes.value: SAMEA4515692
attributes.tag: INSDC center alias
attributes.value: CRUK-CI University of Cambridge
attributes.tag: INSDC center name
attributes.value: CRUK-CI University of Cambridge
attributes.tag: INSDC first public
attributes.value: 2017-02-24T17:02:15Z
attributes.tag: INSDC last update
attributes.value: 2016-10-17T17:12:04Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-5171:Margin BS-seq part 1
attributes.tag: age
attributes.value: 71
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: clinical information
attributes.value: Surgical sample taken from the non-enhancing margin
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: normal
attributes.tag: organism part
attributes.value: brain
attributes.tag: sample name
attributes.value: E-MTAB-5171:Margin BS-seq part 1
attributes.tag: sex
attributes.value: female