title: GSM2111307: E14_d0_H3K27me3_ChIP-seq; Mus musculus; ChIP-Seq
library_construction_protocol: Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. DNA products for ChIP-seq are immuno-precipitated by antibodies of H3K27me3(Millipore, 07-449),H2AZ(Abcam, ab4174). MRE-seq was performed as described (NATURE| Vol 466|8 July 2010). DNA product for 5-hmC_ChIP-seq is generated by a selctive chemical labeling method (Nat. Methods 2011, 9, 75-77). Four parallel digests were performed (HpaII, Hin6I and SsiI are from Fermentas; HpyCH4IV is from NEB), each with 1 ug of DNA. Five units of enzyme per microgram DNA were added and incubated at 37°C for 3 hrs. A second dose of five units-enzyme was added and the DNA was incubated for an additional 3 hrs. Digested DNA was precipitated with sodium acetate and ethanol, and 500 ng of each digest were combined into one tube.  Combined DNA was size-selected by electrophoresis on a 1% agarose gel. A 100-500 bp gel slice was excised and gel-purified using Qiagen Qiaquick columns, eluting in 30 ul of Qiagen EB buffer. Library construction was performed using the Illumina Genomic DNA Sample kit with paired-end adapters, following the manufacturer’s instructions with the following changes. For the end repair reaction, T4 DNA polymerase and T4 polynucleotide kinase were excluded and the klenow DNA polymerase was diluted 1:5 in water and 1 ul used per reaction. For paired-end oligo adapter ligation, adapters were diluted 1:10 in water and 1 ul used per reaction. After the second size selection, DNA was eluted in 36 ul EB buffer using Qiagen Qiaquick columns, and 12 ul used as template for PCR, using Illumina reagents and cycling conditions with 15 cycles. After cleanup with Qiagen MinElute columns, each library is examined by Quant-iT™ dsDNA HS Assay Kit (Qubit, Invitrogen, USA) and Agilent DNA Biaanalyzer (Agilent, USA). The libraries were sequenced with an Illunima Hi-seq Sequencing Systems. MeDIP-seq was performed as described (NATURE| Vol 466|8 July 2010). 3 ug DNA isolated as described above was sonicated to ,100–500 bp with a Bioruptor sonicator (Diagenode). Sonicated DNA was end-repaired, A-tailed, and ligated to paired-end adapters following the standard Illumina protocol. After agarose size-selection to remove unligated adapters, 1 ug of adaptor-ligated DNA was used for each immunoprecipitation using a mouse monoclonal anti-methylcytidine antibody (1 mg/ml, Eurogentec). For this, DNA was heat-denatured at 95 °C for 10 min, rapidly cooled on ice, and immunoprecipitated with 1 ul primary antibody per microgram of DNA overnight at 4 °C with rocking agitation in 500 ml immunoprecipitation buffer (10mMsodium phosphate buffer, pH 7.0, 140mMNaCl, 0.05% Triton X-100). To recover the immunoabsorbed DNA fragments, 2ul of rabbit anti-mouse IgG secondary antibody (2.5 mg/ml, Jackson Immunoresearch) and 100 ml Protein A/G beads (Pierce Biotechnology) were added and incubated for an additional 2 h at 4 °C with agitation. After immunoprecipitation a total of six to ten immunoprecipitation washes were performed with ice-cold immunoprecipitation buffer. A nonspecific mouse IgG immunoprecipitation (Jackson Immunoresearch) was performed in parallel to methyl DNA immunoprecipitation as a negative control. Washed beads were resuspended in TE buffer with 0.25% SDS and 0.25 mg/ml proteinase K for 2 h at 55 °C and then allowed to cool to room temperature. MeDIP and supernatant DNA were purified using Qiagen Qiaquic columns and eluted in 30 ul EB (Qiagen). Twelve cycles of PCR were performed on 10 ul of the immunoprecipitated DNA using the paired-end Illumina PCR primers. The resulting reactions were purified with Qiagen MinElute columns, after which a final size selection (192–592 bp) was performed by electrophoresis in 2% agarose. Libraries were quality controlled by Qubit and Agilent DNA Bioanalyzer analysis. An aliquot of each library was diluted in EB to 5 ng/ul and 1 ul used as template in four independent PCR reactions to confirm enrichment for methylated and de-enrichment for unmethylated sequences, compared to 5 ng of input (sonicated DNA). Two positive controls (SNRPN and MAGEA1 promoters) and two negative controls (a CpG-less sequence on chromosome 15 and GAPDH promoter) were amplified (Supplementary). Cycling was 95 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s with 30 cycles. PCR products were visualized by 1.5% agarose gel electrophoresis.