ES cells and ES-derived neural cells were cultured as described previously11 (link),25 (link). Primary tissues were isolated from 4–6-week-old male 129SvJae/C57/B6 mice. Mouse embryonic fibroblasts (MEFs) and primary neural precursors were isolated from 129SvJae/C57/B6 E14.5 embryos.
RRBS libraries were prepared from 1–10 μg mouse genomic DNA digested with 10–100 Units MspI (NEB). Size-selected MspI fragments (40–120 bp and 120–220 bp) were filled in and 3′-terminal-A extended, extracted with phenol and precipitated with ethanol. Ligation to pre-annealed adapters containing 5′-methyl-cytosine instead of cytosine (Illumina) was performed using the Illumina DNA preparation kit and protocol. QIAquick (Qiagen) cleaned-up, adaptor-ligated fragments were bisulphite-treated using the EpiTect Bisulphite Kit (Qiagen). Preparative-scale PCR was performed and QIAquick-purified PCR products were subjected to a final size selection on a 4% NuSieve 3:1 agarose gel. SYBR-green-stained gel slices containing adaptor-ligated fragments of 130–210 bp or 210–310 bp in size were excised. Library material was recovered from the gel (QIAquick) and sequenced on an Illumina 1G genome analyser.
Sequence reads from bisulphite-treated Solexa libraries were identified using standard Illumina base-calling software and then analysed using a custom computational pipeline. ChIP-Seq experiments, sequencing, alignments and identification of significantly enriched regions were carried out as described previously11 (link).
RRBS libraries were prepared from 1–10 μg mouse genomic DNA digested with 10–100 Units MspI (NEB). Size-selected MspI fragments (40–120 bp and 120–220 bp) were filled in and 3′-terminal-A extended, extracted with phenol and precipitated with ethanol. Ligation to pre-annealed adapters containing 5′-methyl-cytosine instead of cytosine (Illumina) was performed using the Illumina DNA preparation kit and protocol. QIAquick (Qiagen) cleaned-up, adaptor-ligated fragments were bisulphite-treated using the EpiTect Bisulphite Kit (Qiagen). Preparative-scale PCR was performed and QIAquick-purified PCR products were subjected to a final size selection on a 4% NuSieve 3:1 agarose gel. SYBR-green-stained gel slices containing adaptor-ligated fragments of 130–210 bp or 210–310 bp in size were excised. Library material was recovered from the gel (QIAquick) and sequenced on an Illumina 1G genome analyser.
Sequence reads from bisulphite-treated Solexa libraries were identified using standard Illumina base-calling software and then analysed using a custom computational pipeline. ChIP-Seq experiments, sequencing, alignments and identification of significantly enriched regions were carried out as described previously11 (link).