title: GSM3756686: TBP-ChEC Nrd1-AA_drpd3_-Rap_rep2; Saccharomyces cerevisiae; OTHER
library_construction_protocol: The experiment was performed as described in (Zentner et al., 2015) after some modifications. The yeast strains were cultured the day before in YEPD medium. They were diluted to OD600 of 0.2 in YEPD medium in the morning. The 60 mins rapamycin treatment was performed at OD600 of 0.4. For each condition, 50 mL cell culture was harvested at room temperature.  The cells were washed twice in 1 mL of Buffer A (15 mM Tris-HCl pH 7.5, 80 mM KCl, 0.1 mM EGTA, 0.2 mM spermine, 0.5 mM spermidine and protease inhibitor tablet (Sigma-Aldrich). Cells were resuspended in 594 uL of buffer A and 6 uL of 10% digitonin (0.1% final concentration) were added to permeabilize the cells during 5min at 30 °C. Then CaCl2 was added (final concentration 5 mM) to activate the MNase cleavage. 200 uL were collected at 30s for SPT15-MNase or 20s for STH1-MNase and were immediately mixed with 200 uL of 2X Stop solution (400 mM NaCl, 20 mM EDTA, 4 mM EGTA, 1% SDS). The cells were treated with Proteinase K (0.4 ug/uL final concentration) and incubated at 55 °C for 30 minutes. The DNA was extracted using phenol:chloroform:isoamyl extraction, followed by precipitation by adding 30 ug glycogen, 500 uL of 100% ethanol and incubated at -20 °C for one hour. RNAse treatment was performed by adding 34.5 uL of Tris, pH 8.0 and 10 ug of RNAse per reaction. For size selection of DNA fragments, 75 uL of solid phase reversible immobilization beads (SPRI, AmpureXP Beckman Coulter) were added in 25 uL of RNAse treated DNA and the reaction was mixed by pipetting up and down 10 times. The beads were incubated at room temperature for 5 minutes. The tubes were placed in the magnetic rack and supernatant was transferred to the new tube containing 10 mM Tris and 0.2 M NaCl for each reaction. The DNA was extracted with phenol:chloroform:isoamyl solution and precipitated with 100% ethanol. The pellets were washed with 70% ethanol and resuspended in 29 uL of Tris, pH 8.0. DNA concentration was measured using Qubit dsDNA HS assay kit (Invitrogen) using 4 uL sample. The rest 25 uL was used to prepare sequencing libraries using NEBnext Ultra DNA library prep kit for Illumina (NEB). The samples were sequenced in the paired-end mode at the iGE3 genomics sequencing platform in Geneva. NEBnext Ultra DNA library prep kit for Illumina (NEB)
attributes.tag: GEO Accession
attributes.value: GSM3756686