title: GSM3082081: Med14-AID + DMSO TFIIB ChIP-seq; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: After DMSO or 3-IAA treatment, cultures were fixed with formaldehyde (1% final) for 10 minutes at room temperature (RT), then quenched with glycine (125 mM final) for 5 minutes. After washing with PBS, fixed cell pellets were preserved at -80°C. Thawed pellets were later spheroplasted by resuspension in PBS along with 100 μl of 2 mg/mL zymolyase for 5 m at 37°C. All ChIP solutions henceforth were supplemented with a protease inhibitor cocktail. After washing with PBS, spheroplasts were lysed in 150 μL lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10 mM EDTA, 1% SDS) at RT for 5 m. Lysates were then adjusted to 1.5 mL with 1350 μL of dilution buffer (20 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) and transferred to a Covaris milliTube on ice. Sonication of diluted ChIP lysate was performed in a Covaris S220 sonicator, with parameters of 150 W Peak Power, 40% Duty Cycle, and 200 Cycles/burst, for a time of 180 s. After sonication, samples were centrifuged for 10 m at 21,000 x g and 4°C. The soluble lysate was then transferred to a new tube along with 20 μL of Sigma FLAG beads (M8823) pre-blocked with BSA, and the IP was performed for 1 h at RT. Beads were then washed 3 x 1 mL cold wash buffer (20 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% Triton, 0.1% Nonidet-P40) at RT, with each wash lasting 3 m. For extraction and de-crosslinking, washed beads were resuspended in 250 μL elution buffer (50 mM Tris pH 8.0, 250 mM NaCl, 10 mM EDTA, 1% SDS) with 60 μg proteinase K incubated overnight at 1,000 RPM and 65°C in an Eppendorf ThermoMixer. The next morning, ChIP DNA was extracted with 200 μL phenol/chloroform/isoamyl alcohol, treated with 10 μg RNase A for 20 m at 37C, re-extracted with phenol/chloroform, and precipitated with linear acrylamide in >80% EtOH at -80C overnight. Precipitated DNA was pelleted for 20 m at 4°C and 21,000 x g, washed with RT 75% ethanol, re-spun for 5 m, dried at RT, and finally resuspended in 20 μL low-TE (50 mM Tris, 0.1 mM EDTA). Libraries were constructed using the NEBNext Ultra II Library Prep Kit for Illumina at the Indiana University Center for Genomics and Bioinformatics.
attributes.tag: GEO Accession
attributes.value: GSM3082081