title: GSM4462622: BM1247-MDS; Homo sapiens; OTHER
library_construction_protocol: DNA were purified from bone marrow aspirates in corresponding individual person using Qiagen Dneasy Blood and Tissue kit. sCMS-IP-seq was performed as described previously with some modifications. Briefly, genomic DNA was combined with unmethylated λ-bacteriophage DNA (NEB) for bisulfite conversion efficiency check and then sheared into median 300-bp size range using a Covaris M220 Focused-ultrasonicator (Covaris). Sheared DNA was bisulfite converted using a EZ DNA methylation-lightning kit (Zymo Research) with suggested protocols to convert 5-hydroxymethylcytosin (5hmC) to cytosine-5-methylenesulfonate (CMS). CMS-containing DNA fragments were then immunoprecipitated using CMS-specific antiserum pre-conjugated with protein A/G dynabeads. Precipitated DNA fragments were purified by the conventional phenol/chloroform/isomyl-alcohol method. Purified enriched fragments were amplified with random primers followed by adaptor ligation. Final amplification was performed with illumina TruSeq indices using a Pico Methyl-Seq Library Prep Kit (Zymo Research) with manufacturer's protocols. Constructed library size distribution was determined by a Bioanalyzer with an Agilent High Sensitivity DNA Kit (Agilent). Library concentration was measured by a Qubit 4 Fluorometer using a Qubit dsDNA high sensitivity assay kit (Thermo Fisher Scientific). Pooled DNA libraries were sequenced on NextSeq 500 (Illumina) with a NextSeq 500/550 High Output Kit v2 (single-end reads, 75 Cycles) following protocols suggested by the manufacturer (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM4462622