title: GSM4142671: MeCP2_WT_IgG_ChIPseq_R2; Mus musculus; ChIP-Seq
library_construction_protocol: Forebrain (cortex and hippocampus) was dissected from 8-to-12-week-old male mice and flash frozen. Tissue was homogenized in 1% formaldehyde and cross-linked for 10 minutes at room temperature. Cross-linking was quenched with 125 mM glycine for 5 minutes at room temperature. Tissue was pelleted by spinning at 500 g for 5 minutes at 4°C, washed once with PBS, then spun again at 500 g for 5 minutes at 4°C. Cells were lysed by resuspending in L1 buffer (50 mM Hepes pH 7.5, 140mM NaCl, 1 mM EDTA pH 8, 1 mM EGTA pH 8, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, protease inhibitors) and rotating at 4°C for 10 minutes, then spun at 500 g for 5 minutes at 4°C. Nuclei were washed for 10 minutes at 4°C in L2 buffer (10 mM Tris pH 8, 200 mM NaCl, protease inhibitors), then spun at 500 g for 5 minutes at 4°C. Nuclei were resuspended in LB3 buffer (10 mM Tris pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-Lauroylsarcosine, protease inhibitors), and sonicated in a Diagenode Bioruptor (High Power, 60 cycles, 30 seconds on/45 seconds off). Insoluble material was removed by spinning at 16,000 g for 10 minutes at 4°C, and Triton X-100 was added to soluble chromatin at a final concentration of 1%. Chromatin was pre-cleared for two hours with Protein A (for rabbit antibodies) or Protein G (for mouse antibodies) Dynabeads, then incubated with Protein A or Protein G Dynabeads conjugated to antibodies overnight at 4°C. Antibodies used were: H3K27ac (Abcam ab4729), H3K36me3 (Abcam ab9050), H4K12ac (Millipore 07-595), H3K9ac (Millipore 06-942), H3K79me2 (Abcam ab3594), Pol II (Abcam ab817), Pol II Ser5p (Millipore 04-1572), and MeCP2 Ab1 and pre-immune serum (Chen et al., 2003), and MeCP2 Ab2 (Sigma M6818). Beads were washed twice with Low Salt Buffer (20 mM Tris pH 8, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), twice with High Salt Buffer (20 mM Tris pH 8, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), twice with LiCl Wash Buffer (10 mM Tris pH 8, 1 mM EDTA, 1% NP-40, 250 mM LiCl, 1% sodium deoxycholate) and once with TE Buffer (50 mM Tris pH 8, 10 mM EDTA) at 4°C. Chromatin was eluted off beads by incubating in TE Buffer with 1% SDS at 65°C for one hour, and crosslinks were reversed by incubating overnight at 65°C. Chromatin was treated with RNase A for 30 minutes at 37°C and Proteinase K for 2 hours at 55°C. DNA was phenol-chloroform extracted and purified with the Qiagen PCR purification kit. Libraries were generated using the NuGEN Ovation Ultralow System V2 following manufacturer instructions.
attributes.tag: GEO Accession
attributes.value: GSM4142671