GSM3683299: Mbd2_ChIP-seq; Mus musculus; ChIP-Seq
mice (10-12 weeks of age) were sacrificed, the brains were rapidly removed, and bi-lateral hippocampi were isolated, flesh frozen and stored at -80°C for later analysis. hippocampi (n=8) were pooled, homogenized in 1 X PBS including 1% formaldehyde, and the homogenates were kept for 10 min at 25°C. Cross-linking reactions were stopped by the addition of glycine (125 mM) for 10 min at 25°C. Fixed chromatin samples were then homogenized in cell lysis solution (PIPES 5 mM (pH 8), KCl 85 mM, NP40 0.5%) and centrifuged for 5 min at 3000 rpm, 4°C. Pellets were resuspended in RIPA-light solution (NaCl 150 mM, SDS 0.3%, Tris-HCl 50 mM (pH 8)) and sonicated using a Covaris E220. Sonicated chromatin samples were then centrifuged for 15 min at 14000 rpm, 4°C. Pellets were resuspended in 1 ml of RIPA-light solution. Chromatin samples were pre-cleared with 50 µl of Dynabeads protein G (Life Technologies, Ottawa, ON) pre-blocked with BSA and incubated overnight at 4°C with an anti- Mbd2 (Epigentek-A1007, Burlington, ON) antibody. Input control was treated identically the same way except for not adding an anti-Mbd2 antibody to the solution. Antibodies and chromatin were then mixed with 100 µl of Dynabeads protein G for 3 hours at 4°C. The beads were then washed with RIPA-light solution, then with wash-solution (Tris-HCl 100 mM (pH 8), LiCl 500 mM). Protein–DNA complexes were eluted from the beads, de-cross-linked, treated with proteinase K and purified. The DNA concentration was determined by fluorometry on the Qubit system (Invitrogen, Ottawa, ON). A total of 10–12 ng DNA were used for the preparation of the libraries. The immunoprecipitated DNA and input DNA were sheared a second time with the Covaris E220 instrument in 53 µl reaction volume (duty factor 10%, Pic Incident Power 175, Cycles per burst 200, time 360 sec) to obtain fragments in the size range of 150 bp followed by purification with AMPure XP beads (×1.8v/v) (Beckman Coulter A63881, Indianapolis, IN). Purified DNA was resuspended in 45 µl elution buffer. Libraries of the chromatin immunoprecipitated and input DNA fragments were prepared using the Tru Seq DNA Low Throughput Protocol (Illumina). PCR enrichment of ligation products was performed using the Illumina Primer Cocktail; 15 cycles of PCR were performed for ChIP libraries and 10 cycles for the input. The libraries were purified using AMPure XP beads ×1.0 v/v. Quality of libraries was validated by 260 nm absorbance measurement, quality control on HSdna chip (Agilent Bioanalyzer: size of libraries around 275bp bp) and quantification by Q-PCR with Kappa Library Quantification kit for Illumina Sequencing Platforms (KAPPA Biosystems). The DNA concentration of the different sequencing libraries was from 40 to 500 nM. Clusters (13.5 pM) were generated using TruSeq PE Cluster Kit v3, for cBot protocol.
GEO Accession
GSM3683299