ChIP-seq of Saccharomyces cerevisiae: [ESI+] snt1, rep1
Chromatin isolation was performed using a protocol adapted from Kuras and Struhl47. One-half litre of diploid cells were grown to mid-exponential phase per ChIP to be performed, and then fixed with 1% formaldehyde for 1 hr before collection by centrifugation. Cells were resuspended in FA lysis buffer (50 mM HEPE-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, 1% triton X-100, 1X Roche Complete Protease Inhibitors) with 0.5% SDS added, and then lysed using acid-washed glass beads for 15 min at 50 Hz. Chromatin was then precipitated by ultracentrifugation at 237,000 x g for 30 min, then resuspended in 1 ml FA lysis buffer with 0.1% SDS. Chromatin suspension was then fragmented to predominant final DNA length of ~150-200 bp in a Covaris E220 ultrasonicator, and then clarified by centrifugation at 20,000 x g for 30 min. 10% of the resultant chromatin preparations were reserved as input controls. Pan-acetyl H3 (H3ac), pan-acetyl H4 (H4ac), and RNA Pol II ChIPs were performed using antibody-coupled Protein G Dynabeads (antibodies used, respectively: Upstate 06-599, Upstate 06-866, and Active Motif 39097). After precipitation overnight in FA buffer containing 0.1% SDS and 275 mM NaCl, H3ac and H4ac precipitations were washed once with FA buffer containing 0.1% SDS and 500 mM NaCl, once with buffer containing 0.1% SDS and 750 mM NaCl, once with wash buffer (10 mM Tris-HCl, pH 8.0, 0.25 M LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate), and once with TE before being eluted in TE with 1% SDS at 65 C. Snt1-myc, Rap1-myc, and Hos2-myc precipitations were all performed with c-Myc affinity agarose (Sigma-Aldrich A7470) blocked with 2% BSA. After precipitation overnight in FA buffer with 0.1% SDS and 275 mM NaCl, all were washed once with FA buffer with 0.1% SDS and 275 mM NaCl, once with the same buffer but with 500 mM NaCl, one with wash buffer, and once with TE before being eluted in TE with 1% SDS at 65 C. After elution, proteinase K was added (final concentration 0.2 mg/ml) and incubated at 37 C for 2 hrs before crosslink reversal at 65 C overnight. Precipitants were then cleaned up using a standard phenol:chloroform:isoamyl alcohol extraction followed by isopropanol precipitation overnight. DNA was then resuspended in DEPC-treated water, and then the concentration was measured by Qubit spectrophotometer. Libraries for sequencing were then generated using the NEBNext Ultra II DNA Library Prep Kit, and sequencing was performed on the Illumina HiSeq 4000 platform at the Stanford Functional Genomics Facility.