GSM5381254: C1444_cerebrospnial_fluid_from_pediatric_medullablastoma_patient_WGBS; Homo sapiens; Bisulfite-Seq
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 SWIFT Accel-NGS Methyl-Seq DNA Library Kit (Swift Biosciences). Briefly, adaptase was applied to the bisulfite-converted DNA, with adaptors as well as a low-complexity tail simultaneously ligated to the 3′ end of the DNA fragments. Following a primer extension, the bottom strand DNA was synthesized and the 3′ end was ligated with adaptor sequences. ctDNA was mixed with 0.1% unmethylated λ-bacteriophage DNA (NEB) .  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.   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 Methyl-Seq Combinatorial Dual Indexing Kit (Swift Biosciences). DNA library sizes and concentrations were verified by Bioanalyzer. The DNA libraries were then sequenced in Novaseq S4 (pair-end reads, 150 Cycles) following the manufacturer's protocols.