GSM1517779: HCT116-WT-5-mC-PGM; Homo sapiens; MeDIP-Seq
Genomic DNA (1 μg) was isolated from cultured cells and postmortem brain tissue using  the AllPrep DNA/RNA kit (Qiagen) according to the manufacturer’s recommendations for  cells and tissues. DNA concentration was determined using the Qubit DNA Broad Range  fluorescence assay (Life Technologies) and Qubit Instrument (Life Technologies). MeDIP-Seq libraries for Ion Torrent semiconductor sequencing on the Ion Torrent PGM  or Ion Torrent Proton instruments were generated using a modified protocol consisting of  Life Technologies’ Ion Plus Fragment Library kit (Catalog # 4471252) in combination  with a commercially available 5-methylcytosine (MagMeDIP Kit, Catalog # mc-magme-  048) or 5-hydroxymethylcytosine (5hmC Kit, Catalog # AF-110-0016)  immunoprecipitation kit (Diagenode). A nonspecific mouse IgG was used for an  immunoprecipitation reaction as a negative control. For both 5-mC and 5-hmC MeDIPSeq,  1 ug of DNA was sheared to a mean fragment size of 300 bp by sonication with the  Covaris M220 Focused-ultrasonicator instrument (Covaris) in a 50 μl microTUBE AFA  Fiber Screw-CAP (Covaris). Sonicated DNA was end-repaired, purified with Agencourt  AMPure XP reagent beads (Beckman Coulter), and ligated with Ion Torrent compatible  sequencing adapters following the standard Ion Plus Fragment Library Kit protocol.  Adapter-ligated DNA was purified using Agencourt AMPure XP reagent beads and  immunoprecipitated using either a 5-methylcytosine kit (MagMeDIP) or 5-  hydroxymethylcytosine (hMeDIP) MeDIP kit according to the manufacturer’s protocol.  Following either 5-mC or 5-hmC MeDIP, libraries were size-selected from 200-400 bp on  a 2% agarose E-gel (Invitrogen) and extracted using a MinElute Gel DNA extraction kit  (Qiagen) according to the manufacturer’s instructions. Libraries were then amplified at  18 cycles of PCR (95°C, 15 s; 58°C, 15 s; 70°C, 1 min), purified using MinElute columns  (Qiagen), and eluted in 16 μl EB (Qiagen). Libraries were assessed for quality and  concentration on an Agilent Bioanalyzer instrument. An aliquot of each library was used  to assess enrichment efficiency for control methylated DNA and hydroxymethylated DNA  compared to 10% input DNA.
GEO
GSM1517779
GEO Accession
GSM1517779