Protocols

Myers Lab RRBS Protocol (Edited 06-2010)

Reduced Representation Bisulfite Sequencing (RRBS) is used to measure methylation status at more than 500,000 CpG dinucleotides in the genome. Genomic DNA is digested with the methyl-insensitive restriction enzyme Msp1, small fragments are purified by gel electrophoresis, and they are then used to construct an Illumina sequencing library that is treated with bisulfite and amplified by PCR to convert every unmethylated cytosine to a thymidine. The sequenced fragments are aligned to a customized reference genome sequence and used to calculate the number of reads at each CpG assayed and the percentage of those reads that detect methylation. 

Myers Lab ChIP-seq Protocol v041610

Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) is used to identify transcription factor binding sites in the genome.  Transcription factors (TF) bound to genomic DNA are crosslinked to preserve these interactions, the DNA is fragmented and the DNA::TF complexes are immunoprecipitated (IP) using an antibody to the TF.  The crosslinks are reversed and the DNA fragments are used to make and Illumina sequencing library that is then sequenced on the GAIIx.  The sequenced fragments are compared to a background sequencing library with a peak-calling algorithm to identify DNA sequences enriched by IP that are likely bound by the transcription factor or a complex that includes the immunoprecipitated TF.