title: GSM3223353: MeDIP.5mC.d0; Mus musculus; MeDIP-Seq
library_construction_protocol: Genomic DNA from FACS-sorted cells was randomly sheared to 100-500 bp in a microtube (Covaris; 520045) using a Covaris S220 device (400 sec; 4°C; PP 140; DF 10; CB 200). Sonicated DNA was end-repaired, A-tailed, and ligated to Illumina multiplex adaptors according to NEBNext DNA library prep kit (NEB E6040S). Ligated DNA was purified using Agencourt AMPure XP beads (Beckman Coulter). 1 µg of adaptor-ligated DNA was used for each immunoprecipitation and heat-denatured at 95°C for 10min, rapidly cooled on ice and immunoprecipitated overnight at 4 ˚C with rocking agitation in 500 ml immunoprecipitation buffer (10mM sodium phosphate buffer, pH 7.0, 140mM NaCl, 0.05% Triton X-100) using 1 µl of mouse monoclonal anti-5-methylcytosine antibody (Eurogentec BI-MECY-0100) or 0.5 µl of rabbit 5-Hydroxymethylcytosine antibody (Active motif 39769). To recover the antibody-bound DNA fragments, 50µl Protein G Dynabeads (ThermoFisher) and, only to anti-5-methylcytosine IPs, 5µl of rabbit anti-mouse IgG secondary antibody (Active Motif cat.53017) were added and incubated for an additional 2 h at 4 ˚C with agitation. After immunoprecipitation, a total of seven to ten immunoprecipitation washes were performed with ice-cold immunoprecipitation buffer. Washed beads were resuspended in TE buffer with 0.25% SDS and 0.25 mg/ml proteinase K for 2 h at 55 ˚C with vigorous shaking (900 rpm). DNA was purified with the Qiagen PCR clean-up MinElute kit (Qiagen) and eluted in 30 ul. Samples were then amplified by PCR with Illumina primers (NEBNext Multiplex Oligos for Illumina cat. E7335) in a 50 μl reaction with 2× PCR master mix (NEB cat. M0541). PCR cycled as: (1) 98 °C, 30s; (2) 98 °C, 10 s; (3) 60 °C, 30 s; (4) 72 °C, 30s; (5) repeat steps (2)–(4) for 4-10 cycles; (6) 72°C, 5min. Amplified libraries were purified using Agencourt AMPure XP beads (Beckman Coulter). Quality control was carried out with a Qubit fluorometer and a Bioanalyzer (Agilent). NEBNext DNA library prep kit (NEB E6040S).
attributes.tag: GEO Accession
attributes.value: GSM3223353