GSM5115453: Spo_AI_1; Saccharomyces cerevisiae; OTHER
Nuclei were isolated from yeast cells by grind well in liquid nitrogen, followed by SDS (final concentration: 0.5%) /proteinase K (final concentration: 0.1 mg/ml) treatment at 37 °C overnight. Add 1/4 volume 5 M KAc, mix and place the tube on ice, 10~20 min. Genomic DNA was extracted by the phenol-chloroform method and precipitated with isopropanol. DNA fragmentation was performed at 37 °C overnight using endonucleases (Alu I, DdeI, MboI, MesI and Rsa I; New England Biolabs, Ipswich, MA, USA); the negative control was treated with RNase H (New England Biolabs) at 37 °C overnight before endonucleases treatment. DRIP was performed as described previously (Xu et al., 2017). Briefly, 3.5 ug of DNA was bound with 10 ug of S9.6 antibody (ATCC, Manassas, VA, USA) in 1 X binding buffer (10 mM NaPO4 pH 7, 140 mM NaCl, 0.05% Triton X-100) overnight at 4˚C. 50 ul Protein G sepharose beads were added for 4 hours. Bound beads were washed 4 times in binding buffer and elution was performed in elution buffer (50 mM Tris pH 8, 10 mM EDTA, Proteinase K) for 45 min at 55˚C. DNA was purified and sonicated to 150 bp fragments. The DRIPed DNA samples were sonicated to a set size of 250 bp with an M220 Focused-ultra sonicator (Covaris, Woburn, MA, USA). The sonicated DNA was denatured at 95 °C for 2 minutes and immediately placed on ice for 2 minutes to obtain ssDNA fragments. The first adapter (Adp1, GATCGGAAGAGCACACGTCTGAACTCCAGTCAC(i7)ATCTCGTATGCCGTCTTCTGCTTG) was ligated to the 3' end of the ssDNA using Adaptase (Swif Biosciences, Ann Arbor, MI, USA) via a highly efficient, proprietary reaction that tails only the 3' ends of ssDNA and ligates the first truncated adapter to the 3' ends simultaneously. This method avoids the bias inherent in random primer-based methods, as it ligates adapters in a sequence independent manner. The extension step was performed using the primer paired to the first adapter, followed by a ligation reaction to add the second truncated adapter (Adp2, AATGATACGGCGACCACCGAGATCTACAC(i5)ACACTCTTTCCCTACACGACGCTCTTCCGATCT) to the 5' ends. An indexing PCR step was performed to add the indexed sequence, and the library was amplified. The libraries were checked on an Agilent (Palo Alto, CA, USA) BioAnalyzer, followed by sequencing on an Illumina (San Diego, CA, USA) HiSeq X 10 system.