GSM7890716: Rtt101-HA_ChIP_G1_rep1; Saccharomyces cerevisiae; ChIP-Seq
Cells were crosslinked with 1 % formaldehyde for 15 min at room temperature, quenched with 250 mM glycine incubated for 5 min, followed by ice-cooling for at least 10min. Before freezing, pellets were washed twice with cold 1X Phosphate Buffer Saline (PBS) and transferred into 2 mL screw-cap tubes. All subsequent steps were performed at 4°C or on ice, if not mentioned otherwise. Frozen pellets were resuspended in 1 mL cold FA lysis buffer (50 mM HEPEs-KOH pH 7.5; 140 mM NaCl; 1 mM EDTA; 1 % Triton X-100; 0.1 % sodium deoxycholate; protease inhibitor cocktail) and lysed by MagNA Lyzer (6000 rpm; 5 cycles of 30 secs; 1 min on ice between runs). Extracts were recovered in a new tube and centrifuged at 13,000 x rpm for 30min. After resuspension with 1 mL of fresh FA lysis buffer, extracts were sonicated via Bioruptor Twin (Diagenode) at 4°C for 20 cycles of 30 secs. After centrifugation at 13,000 x rpm for 15 min, supernatant was transferred in a new tube and the amount of protein was determine by Bio-Rad protein assay (cat. 500-0006).  Each immunoprecipitation was carried out with 1 mg of protein (1/10 retained as the input) incubated overnight at 4°C under rotation, with 1 uL of anti-HA antibody. Protein G sepharose beads were then added for 3 hours, at 4°C under rotation.  After incubation, beads were washed 1x with 500 µL cold FA lysis buffer, 2x with 500 µL cold FA-500 buffer (FA lysis with 500 mM NaCl), 2x with 500 µL Buffer 3 (10 mM Tris-HCl pH 8; 1 mM EDTA; 250 mM LiCl, 1 % IGEPAL; 1 % sodium deoxycholate) and 1x with 500 µL 1X TE (50 mM Tris-HCl pH 7.5; 10 mM EDTA). Elution was then performed twice with 100 µL elution buffer (50 mM Tris-HCl pH 7.5; 1 % SDS; 10 mM EDTA) incubated at 65°C for 8 minutes. Eluted sample and input were treated with 0.75 mg/mL Proteinase K and incubated at 42°C for 2 hours, followed by de-crosslinking overnight at 65°C.  Both IP and input were purified with the MinElute PCR Purification kit (Qiagen, cat. 28006), following manufacturer's recommendations and eluted from the column twice with 30 µL of elution buffer from the kit. NEBnext Ultra DNA library prep kit for Illumina (NEB, cat. E7370L)