Genome-wide mapping of ssDNA in mouse genome supercoiled in vitro
Genomic DNA was isolated from a total of 4x10^7 mouse activated B cells by RNase and proteinase K treatment in lysis buffer (10 mM Tris-Cl, pH 8.0, 100 mM EDTA, 0.5% SDS), followed by repetitive phenol:chloroform extraction and ethanol precipitation. Purified DNA was divided in 2 aliquots separately digested with ApoI or Taq1 restriction enzymes. After complete digestion and purification, fragmented DNA samples were separately ligated by T4 DNA ligase at a DNA concentration of 0.4 µg/ml. The efficiency of ligation was analyzed by 2-D agarose gel electrophoresis: circular DNA has faster mobility in the second direction in comparison to linear fragments or nicked circle when chloroquine is present in the running buffer. Linear DNA fragments were subsequently removed by Lambda Exonuclease digestion. After purification, each DNA sample was again split into the two aliquots: samples 1 and 2. Samples 1 were treated with Topoisomerase I as recommended by the supplier. Samples 2 were treated with Topoisomerase I in the presence of ethidium bromide at 8 µg/ml. This topoisomerase-ethidium bromide technique produced topoisomers of circular DNA with an average superhelical density of ‒0.06 ± 0.01 (Singleton and Wells, 1982). Relation between ethidium bromide concentration and superhelical densities of DNA was established in the pilot experiments with plasmid DNA (Kouzine et al., 2004). Relaxed DNA of samples 1 were pooled together as well as supercoiled DNA of samples 2.