title: GSM2188900: MeDIP-seq P19, RA 6hrs; Mus musculus; MeDIP-Seq
library_construction_protocol: For MeDIP and hMeDIP, cells were scraped in phosphate buffered saline and were pelleted at 100 g before genomic DNA extraction using a DNeasy Blood and Tissue kit (Qiagen, France). For ChIP, P19.6 cells were cross-linked with 1.5% formaldehyde for 10 min at room temperature. The cells were rinsed with cold PBS, harvested and lysed with 1% SDS, 10 mM EDTA, and 50 mM Tris-HCl (pH 8.1) containing a protease inhibitor cocktail (Roche). Chromatin fragmentation was subsequently obtained by sonicating samples for 14 min (30 sec on/off cycles) using a Bioruptor (Diagenode) set up at the highest intensity. The soluble chromatin was diluted 10 times in a buffer containing 1% Triton, 2 mM EDTA, 150 mM NaCl, and 20 mM Tris-HCl (pH 8.1), and incubated overnight with antibodies. Sepharose beads (Amersham Pharmacia Biosciences) were then added to the samples together with yeast tRNA as non-specific competitors. After 4 h, beads were serially washed using 1mL of washing buffer I (2mM EDTA, 20mM Tris-HCl (pH8.1), 0.1% SDS, 1% Triton X-100, 150mM NaCl), washing buffer II (2mM EDTA, 20mM Tris-HCl (pH 8.1), 0.1% SDS, 1% Triton X-100, 500 mM NaCl), washing buffer III (1mM EDTA, 10mM Tris-HCl (pH8.1), 1% NP-40, 1% deoxycholate, 0.25M LiCl) and then twice with 1mM EDTA, 10mM Tris-HCl (pH8.1), and immune complexes were eluted using 100 mL of 1% SDS and 0.1 M NaHCO3. Samples were incubated overnight at 65C to reverse cross-linking. DNA was finally purified using the NucleoSpin Extract II kit (Macherey-Nagel). Each ChIP was performed 10 times. The (h)MeDIP, ChIP, FAIRE and input libraries were prepared using the TruSeq ChIP Sample Prep Kit (Illumina, ref. IP-202-1012). Libraries were sequenced by Illumina HiSeq following the manufacturer’s protocol.