Project description:Transcription profiling by high throughput sequencing of maize seedlings, mature pollen, embryo-sac-enriched samples and ovules with embryo sacs removed
Project description:We analyzed levels of 5-methyl cytosine nnnn CCCGGG target sites by sequential restriction digest by SmaI and XmaI enzymes, ligating Illumina adaptors to the restriction fragments and reading methylation-specific signatures at the ends of restriction fragments by paired ends Illumina high throughput sequencing. Digital restriction enzyme analysis of methylation (DREAM) was performed to determine the methylation profile of SW48 colon cancer cell line genomic DNA. Genomic DNA spiked in with unmethylated, partially methylated and fully methylated standards was sequentially cut at CCCGGG sites with the methylation-sensitive enzyme SmaI (blunt ends) and its methylation-tolerant neoschizomer XmaI (5'CCGG overhangs), creating different end sequences that represented methylation status of the CCCGGG sites. These end sequences were analyzed by Illumina high throughput sequencing. Methylation status at individual CCCGGG sites across the genome was determined by counting the methylated reads with the CCGGG signature and unmethylated reads with the GGG signature at the beginnings of the sequencing reads after alignment to the human genome.
