Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: We manually conjugated antibody against cell type markers, such as GFAP, Nestin, Vimentin, PSA-NCAM, or Doublecortin to Dynabeads. We then used the Dynabeads-conjugated antibody to purify cells dissociated from SVZ microdissection. Briefly, cells from fresh dissected baboon SVZ were immediately dissociated with Accutase, equilibrated in binding buffer containing phosphate-buffered saline (PBS), 0.05% TritonX-100 (or saponin, detergent choice depends upon antibody), and subsequently subjected to Dynabeads-conjugated antibody purification. After elution with high salt and pH-gradient buffer, the purified populations were crosslinked in 1.1% formaldehyde before chromatin shearing by Diagenode Bioruptor. The resulting sheared chromatin fragments in a size range between 200 to 500 base pairs were incubated with H3K9me3 antibody-conjugated Protein A Dynabeads (Millipore #07-473, Upstate #07-442; Active Motif #39162; Life Technology Dynabeads protein A) overnight. For normalization, the aliquot of sheared chromatin fragments were incubated with antibody against total histone 3-conjugated Protein A Dynabeads (unmodifiedH3 antibody, Millipore #05- 499; 1:1000). Subsequently, enriched chromatin fragments were eluted, subjected to de-crosslink, purified for library preparation (Illumina Library Kit) and sequenced with 200millinon tags through Illumina HiSeq2000 sequencer. Libraries were prepared according to instructions for Illumina Library Kit, Illumina TruSeq v2