In Methyl-seq, 5 µg genomic DNA was digested with HpaII or MspI. Fragments were made into next-gen sequencing libraries by using adapters and reagents from Illumina's Library Construction Kit. After an initial round of PCR, DNA bands (fragments with adapters) of 100-350 bp were isolated by gel extraction (Supplemental Fig. 2). Size-selected libraries were then PCR-amplified prior to sequencing. For meDIPSeq, 5 µg genomic DNA was digested with MspI and immunoprecipitated with anti-methylcytosine antibody (Calbiochem); fragments were made into Illumina libraries as described above.
Library strategy
OTHER
Library source
genomic
Library selection
other
Instrument model
Illumina Genome Analyzer
Description
please supplementary file GSE14966_sequence_annotations.txt.gz on Series record for sequence information methylation call, Library2, 0=unmethylated (tag average>1), 1=methylated (tag average<=1)
Data processing
Usable sequence reads were mapped to CCGG sites predicted in silico. Sites with four or more MspI tags occurring in either the forward or reverse direction were retained in the analysis. These assayable sites were then grouped with neighboring sites that were within 35-75 bp. Regions therefore represent any number of digestion sites (between 2 and 18) that have neighboring sites within 35-75 bp of each other. Methylation calls were made by using HpaII tag data from all assayable cut sites. The larger of either the forward read count or reverse read count for each site was averaged across each region. Regions that had an average of zero or one reads per digestion site were called methylated, and regions with more than one sequence read per site were considered unmethylated.