GSM8741968: YNN1 replicate 1 60nM Cbf1; synthetic construct; MNase-Seq
Nucleosome binding experiments were performed by incubating 60 nM nucleosome with 0 nM or 60 nM of TFs (Reb1, Cbf1, or Pho4) for 20 minutes at room temperature. Nucleosomes-TF binding was confirmed on a 5% acrylamide native gel. The incubated samples were then digested with 0.3 U, 1 U, 3 U, 10 U of MNase (New England Biolabs) for 8 minutes at 37 ⁰C and immediately quenched by adding quench buffer (0.1 % SDS, 20 mM EDTA pH8.0, 0.01 U proteinase K). Proteinase K digestion was carried out by incubating the quenched samples for 1 hour at 50 ⁰C. The final digested samples were analyzed by agarose gel electrophoresis with a 1.5% gel in 0.5x TAE buffer (20 mM Tris base, 10 mM acetic acid, 0.5 mM EDTA pH 8.0) (Figure S4). The digest DNA samples were then purified by agarose gel electrophoresis and then gel extraction with a Freeze 'N Squeeze DNA extraction kit (Bio-Rad). After gel purification of the MNase digested samples, sequencing libraries were prepared using NEBNext ® Ultra™ II DNA Library Prep Kit for Illumina® (Catalog # E7645L) according to the manufacturer's instructions. The sequencing libraries were sequenced on a NextSeq 2000 machine in 2 x 50 bp paired-end sequencing mode at the Genomics Research Incubator at The Pennsylvania State University.