title: GSM2837727: FLAG_WT; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: Every ChIP-seq cells were grown up to mid-log phase and harvested after final 1% formaldehyde fixation and 125mM glycine quencing. Cells were harvested by centrifugation at 4 Celcius degrees and the cell pellets were washed by 15mL of pH7.5 TBS buffer per a conical tube. ChIP cells were disrupted by glass bead vortexing in ChIP  lysis buffer and sonicated by Sonic Dismembrator Model 500, Fisher Scientific. Debris of the sonicate was excluded by centrifugation by 14,800rpm at 4 Celcius degree. IP samples were aliqouted from the sonicate and immunoprecipiated by proper amount of antibody and protein A/G beads, washed by ChIP  lysis buffer for over-night at 4 Celcius degrees. IP samples were washed and eluted by proper buffers in  Sigmaprep spin column. Proteins in both IP and input samples were degraded by 2hr 30ug proteinase K rxn and de-crosslinked at 65 Celcius degrees for at least 8hrs. The IP and input DNA were eluted by Quiagen PCR purification kit. Total RNA for mRNA-seq was prepared by the hot-phenol extraction. For preparation of MNase-seq library, formaldehyde-fixed cell pellets were resuspended in 10 ml of pre-incubation buffer (20 mM EDTA, 0.7 M mercaptoethanol), washed, and resuspended in 2 ml of zymolyase buffer (1 M sorbitol, 50 mM Tris-Cl, pH 8.0, 5 mM mercaptoethanol) containing 2.5 mg ml-1 zymolyase (20T; US Biological). Lysed cells were spheroplasted at 30°C  for 20 min and chromatin was isolated. Pelleted nuclei were digested with 200 units of MNase (NEB) at 37°C for 20 min (confirmed as a typical digestion condition by comparison of fragment sizes, data not shown), and then reverse cross-linked in 10 mM EDTA and 1% SDS buffer with proteinase K for 6 hours at 37°C. The DNA was extracted with phenol/chloroform, purified by ethanol precipitation, and resolved by 2% agarose gel electrophoresis in TAE buffer. Yeast cells (2.5 million) were harvested at O.D. 0.6~0.8/mL (mid-log phase), and spheroplasted in 200 μl of Sorbitol buffer (1.4 M Sorbitol, 40 mM Tris-HCl, pH 7.5, 0.5 mM MgCl2) with 10 μl of 50 mg ml-1 of zymolyase (20T; US Biological). After washing twice with 100 μl of ice-cold Sorbitol buffer, the spheroplasts were incubated in fresh TD buffer (20 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 20% dimethylformaldehyde) with transposase at 37°C for 4 hours. QIAquick PCR purification kit was used to purify DNA fragments, and transposition reactions and PCR amplifications were performed. General Manual of Nextflex ChIP library preparation kit. mRNA isolation and mRNA-seq library preparation were performed using the NEXTflex Rapid Directional mRNA-Seq Bundle.
attributes.tag: GEO Accession
attributes.value: GSM2837727