title: GSM2102275: Dnmt1KD_hMeDIP_rep1; Mus musculus; MeDIP-Seq
library_construction_protocol: Genomic DNA was isolated from ES cells by resuspending cells in ES cell lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM EDTA, 10 mM NaCL, 0.5% sarkosyl), treating with RNase A, and incubating with 1 μg/μL proteinase K overnight at 55°C. DNA was cleaned through PCI extraction and isopropanol precipitation. Genomic DNA was then diluted to 20 ug/mL in TE buffer and sonicated in a Bioruptor (UCD-200) on low for 20 minutes with intervals of 15 seconds on/15 seconds off at 4°C. Sheared DNA (ranging in size from 200-600 bp) was prepared for deep sequencing and further processing by end-repaired, A-tailed, and adaptor-ligated with DNA purification through PCI extraction and ethanol precipitation between each step. 1.5 μg of prepared DNA was diluted in TE buffer up to 450 μL, denatured for 10 minutes at 95°C and immediately transferred to ice for 10 minutes. Denatured DNA was then incubated with 50 μL 10X DNA IP buffer (100 mM Na-HPO4 pH 7.0, 1.4 M NaCl, 0.5% Triton X-100) and 1.5 μL anti-5hmC (Active Motif 39791) overnight at 4°C with constant rotation. 40 μL of magnetic beads (anti-rabbit IgG for 5hmC, Life Technologies) were pre-blocked with BSA and then resuspended in DNA IP buffer. Beads were then added to the DNA/antibody mixture and incubated for 2 hours at 4°C with constant rotation. Beads were then washed three times with 700 μL 1X DNA IP buffer and DNA was eluted from the beads by incubating at 50°C on a thermomixer with 250 μL 1X DNA IP buffer and 4 μL proteinase K (20 μg/μL) for 3 hours. DNA from IP samples were cleaned through PCI extraction and ethanol precipitation. DNA was PCR amplified with KAPA HiFi polymerase using 16 cycles of PCR. Each library was size-selected on a 1% agarose gel, its concentration determined using a Qubit (Thermo), and the integrity was confirmed by Topo-cloning a portion and sequencing ~10 fragments from each library. Libraries were sequenced on an Illumina HiSeq2000 using single-end sequencing at the UMass Medical School deep sequencing core facility. Resulting fastq sequences were processed and analyzed using the HOMER software.
attributes.tag: GEO Accession
attributes.value: GSM2102275