GSM3333570
GSM3333570: mcm2-3a H3K56ac eSPAN repeat 2; Saccharomyces cerevisiae; ChIP-Seq
All ChIP and eSPAN assays were performed as described previously except that Mnase digestion was used for chromatin fragmentation in order to map nucleosomes. Briefly, the samples were incubated with 1% fresh-made formaldehyde at 25 °C for 20 min, and then quenched with 0.125M glycine for five min.  The cells wer harvested and lysed with glass beads in ChIP lysis buffer (50 mM HEPES, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.1% sodium deoxycholate). The resulting cell and chromatin pellet was washed with NP buffer (1.6 M sorbitol, 2 mM CaCl2, 5mM MgCl, 50 mM NaCl, 14 mM β-mercaptoethanol, 10 mM Tris-HCl (pH 7.4), 0.075% NP-40, 5 mM spermidine).  Proper amounts of Mnase was added to ensure that majority of chromatin are mono- and di-nucleosomes with incubation at 37 °C for 20 min . The reacation was terminated by adding 5ul 0.5M EDTA. Further, ¼ volume 5X ChIP lysis buffer was added into dilute thedigested chromatin. The lysates was incubated in ice for 30 minutes. After clarification of lysates by centrifugation, soluble chromatin (mainly mononucleosomes) was immunoprecipitated with a specific antibody including anti-H3K4me3 antibody (ab8580), anti-H3K56ac antibody (6), anti-HA antibody (12CA5) or anti-T7 (A190-117A BETHYL)). ChIP DNA was recovered using Chelex-100. The ChIP or total DNA was denatured at 100°C for five min and was immediately transferred to ice bath for five minutes.  The DNA was diluted at least ten times with BrdU IP solution (PBS, 0.0625% Triton X-100(v/v), 6.7 g/mL Escherichia. coli tRNA, 0.67 ul/mL BrdU antibody (BD Bioscience)).  After the samples were incubated at 4°C for two hour, 20 uL protein G beads (GE Healthcare) were added.  After incubation at 4°C for another hour, the protein G beads was washed extensively. DNA were eluted using 100 uL 1XTE buffer plus 1% SDS and incubation at 65 °C for 15 min. Eluted DNA was purified using a Qiagen Mini-elute kit. We used the ssDNA library preparation kit to prepare library (Cat no. 10096 Swift Bioscience). The ssDNA libraries were sequenced using the paired-end method by different sequencing platform over the years including Hi-seq 2000 or 2500 machine at Mayo Clinic or Columbia University.
GEO
GSM3333570
GEO Accession
GSM3333570