GSE158336
A compendium of chromatin contact maps reflecting regulation by chromatin remodelers in budding yeast
We employed in situ Hi-C with an auxin-inducible degron (AID) system to demonstrate the effect of chromatin remodeling on 3D genome organization in yeast. Overall design: IAA (Sigma, I2886) was added to a final concentration of 0.5 mM for degradation of target proteins and the same volume of 100% ethanol was used as a control. For G1 arrest, alpha-factor was added at a final concentration of 50 ng/ml to bar1? strains; after 2 h, 0.5 mM IAA was added and the cells were incubated for an additional 3 h. For S or G2/M arrest, alpha-factor was added at a final concentration of 50 ng/ml to bar1? strains; after 1.5 h, the yeast cells were transferred to fresh YPD medium containing 200 mM hydroxyurea (HU; Sigma, H8627) for S arrest or 15 ug/ml nocodazole (Sigma, M1404) for G2 arrest. At 1.5 h after HU/nocodazole treatment, 0.5 mM IAA was added and cells were incubated for an additional 3 h. For G2 arrest, an additional 10 ug/ml of nocodazole was applied along with the IAA. Yeast cells (50 O.D.600) were cultured, fixed with 3% formaldehyde (Wako, 064-00406), quenched, pelleted, pre-incubated with ß-ME buffer (20 mM EDTA and 0.7 M ß-ME) for 10 min at 30?, and then lysed with 2 mg of zymolase (US Biological, Z1004) in 2 ml lyticase buffer (1 M sorbitol, 50 mM Tris-Cl (pH 8.0), 5 mM ß-ME) for 20 min at 30?. The obtained spheroplasts were resuspended in 2 ml of ice-cold PBS. The isolated nuclear DNA was resuspended in 50 ul of 0.5% SDS, incubated for 10 min at 62?, mixed with 170 ul of 1.47% TritonX-100 and incubated for 15 min at 37?. The generated libraries were sequenced using 150-bp paired-end reads on an Illumina Novaseq6000 and/or HiSeqX.