title: Hsf1_t30_0.8U
design: 70 ml of yeast cells were grown in SC at 30oC to an OD595 of 1.0 in three independent biological replicates. 30 minutes before crosslinking, either rapamycin ('30min') to a final concentration of 7.5 µM or the same amount of DMSO ('0min')  was added to the samples. The cells were crosslinked at RT for 5 minutes using formaldehyde at a final concentration of 1% and quenched for 5 minutes with glycine (final concentration = 125 mM). Subsequently the cells were spun down and washed once with sorbitol (1 M) and spheroblasted for 8 minutes using 1 ml of spheroblasting buffer (1 M Sorbitol, 1 mM β-mercaptoethanol, 10 mg/ml Zymolyase 100T (USB)). The spheroblasts were spun down and washed twice before resuspending them in 1 ml of MNase digestion buffer (1 M Sorbitol, 50 mM NaCl, 10 mM Tris pH=7.5, 5 mM MgCl2, 1 mM CaCl2 , 0.075% Nonidet P-40, 1 mM β-mercaptoethanol, 0.5 mM Spermidine). The samples were divided over 4x four-fold different MNase amounts (Sigma-Aldrich): 0.046875U, 0.1875U, 0.75U and 3.0U and incubated at 37oC for 45 minutes. The digestion was stopped by putting the samples on ice and immediately adding 15 µl of 0.5M EDTA and subsequently 15 µl of 10% SDS. The proteins were degraded by adding 15 µl of (10 mg/ml) proteinase K and incubating the samples for 1 hour at 37oC. The crosslinks were reversed by putting the samples at 65oC overnight. The next day nucleosomal DNA was isolated using phenol extraction and RNA was digested using RNase A/T1 (Thermo Scientific; final concentration 0.2 mg/ml & 250 U/ml). Sequencing libraries were created from the purified nucleosomal DNA using the NextflexTM rapid DNA-Seq kit (Bioo Scientific) using a modified protocol.