Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA
Figure Lengend Snippet: Bisulfite footprinting by deep-sequencing across the FMR1 repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.
Article Snippet: For ssDNA displacement at FMR1 , GC-RICH solution was added to the PCR mix, according to the manufacturer’s instructions (Roche).
Techniques: Footprinting, Sequencing, Amplification, Methylation