ChIP-seq was done based on methods established previously (Li, Y et al. 2014). Two independent chromatin immunoprecipitation assays were performed using MAGnify chromatin immunoprecipitation kit (Life Technologies, Grand Island, NY). Specifically, main olfactory neuroepithelia (MOE) were dissected from the nasal cavity and dissociated mechanically via trituration in phosphate buffer saline (PBS). Equal number of cells was used for subsequent steps. Protein-DNA complexes were cross linked by incubating dissociated MOE cells with 1% formaldehyde for 5 minutes on a rocker at room temperature. The cross-linking was quenched by adding Glycine. After washing off the media, MOE cells were lysed briefly in SDS containing buffer followed by sonication for 15 minutes with 30 second intervals to shear genomic DNA using a BioruptorTM 300 (Diagenode, Denville, NJ). Sheared DNA was evaluated by 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and selected for sizes ranging between 200 bp and 300 bp. Each sheared genomic DNA preparation was split into two equal portions and incubated with MeCP2 antibody (Diagenode, pAb-052-050) or Rabbit IgG (Millipore, Cat# 12-370) for the negative control. MeCP2-genomic DNA complexes were pulled down and reverse cross-linked by DNase-free Proteinase K and subsequently purified. MeCP2 ChIP-seq and input DNA library were prepared according to manufacturer’s instruction (Bioo Scientific, Austin, TX) using 5143-01 NEXTflex ChIP-Seq kit and 514120 NEXTflex ChIP-Seq Barcodes-6. ChIP and Input DNAs were PCR amplified, cleaned up and sequenced on Illumina Hi-Seq 2000 at QB3 Vincent J. Coates Genomics Sequencing Laboratory in the UC Berkeley. library construction protocol
GEO Accession
GSM1827604