title: GSM4223618: pediatric medulloblastoma tumr tissue CMS-IP MB2_tumor.CMS; Homo sapiens; OTHER
library_construction_protocol: Total DNA containing cell-free tumor DNA (ctDNA) was isolated from 200-400 µL cerebral spinal fluid (CSF) using QIAamp Circulating Nucleic Acid Kit (Qiagen) according to manufacturer's instructions. For MB tumor tissues, up to 20 mg of tumor tissues was snap frozen with liquid nitrogen and grinded. DNA was then isolated using an AllPrep DNA/RNA mini kit according to the manufacturer's protocol. The isolated DNA concentration was measured by a Qubit 4 Fluorometer with the Qubit dsDNA high sensitivity assay kit (Thermo Fisher Scientific). The whole genome bisulfite sequencing (WGBS) analysis was used to measure the genome-wide DNA methylation status. The ctDNA WGBS libraries were generated using a Pico Methyl-Seq Library Prep Kit (Zymo Research). Briefly, ctDNA was mixed with 0.1% unmethylated λ-bacteriophage DNA (NEB) followed by sodium bisulfite conversion. Then the bisulfite converted DNA was annealed with random primers for initial amplification, followed by adaptor ligation and final amplification with Illumian TruSeq indices. Constructed libraries were run on 2% agarose gel to assess the size distribution and library concentration was measured by a Qubit 4 Fluorometer with a Qubit dsDNA high sensitivity assay kit (Thermo Fisher Scientific). Normalized libraries were then pooled in an equimolar ratio and sequenced on NextSeq 500 (Illumina) with a NextSeq 500/550 High Output Kit v2.5 (single-end reads, 75 Cycles) following the manufacturer's protocols.  CMS-IP-seq was performed as described previously with some modifications. Briefly, purified MB tumor gDNA was sonicated to the 200-400 bp size range using Covaris M220 Focused-ultrasonicator (Covaris). The CSF ctDNA was directly treated with sodium bisulfite without sonication. The sheared gDNA and purified ctDNA were treated with sodium bisulfite using an EZ DNA methylation-lightning kit (Zymo Research) with manufacturer's protocols to convert 5hmC to cytosine-5-methylenesulfonate (CMS). Next, CMS-containing DNA fragments were immunoprecipitated using a CMS-specific antiserum conjugated with protein A/G dynabeads. Enriched DNA fragments were purified using the conventional phenol/chloroform/isomyl-alcohol method, and amplified with random primers, followed by adaptor ligation and final amplification with illumine TruSeq index using a Pico Methyl-Seq Library Prep Kit (Zymo Research). DNA library sizes and concentrations were verified by Bioanalyzer. The DNA libraries were then sequenced in NextSeq 500 (Illumina) with NextSeq 500/550 High Output Kit v2.5 (single-end reads, 75 Cycles) following the manufacturer's protocols. WGBS and CMS-IP
attributes.tag: GEO Accession
attributes.value: GSM4223618