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S. cerevisiae proteome response to heat stress - 240 min time course. C4PR_LIV

ABSTRACT: In this project, we follow the temporal proteome dynamics during heat stress response in a model organism Saccharomyces cerevisiae. Using label-free quantification we determine changes in the yeast proteome at different times after mild temperature shift, from 30˚C to 37˚C. The time points measured were 0 min (30˚C, no stress) and then at 10 min, 30 min, 60 min, 120 min and 240 min after cells were transferred to 37 °C. For each time point, four biological replicates were collected for the haploid wild type (BY4742, Mat ALPHA, his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; YJL088w::kanMX4) strain and a haploid strain with a chaperone Ssb1p deletion (ssb1Δ).

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ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Haploid Cell

SUBMITTER: Andrew Jarnuczak

LAB HEAD: Professor Simon Hubbard

PROVIDER: PXD006262 | Pride | 2018-01-17

REPOSITORIES: Pride

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Project description:2×10^7 HL60 cells were treated with 10 uM ATRA for 2 and 5 days, and conducted Hi-C to study the chromosomal architecture. Cells were fixed by 1% formaldehyde (v/v) for 15 min at room temperature (RT) with slowly rotation. Cells were lysed by 550 μl lysis buffer (500 μl 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630 and 50 μl protease inhibitors) using a homogenizer, and harvested the chromatin at 5,000 rpm and washed twice. The pellet was added 1% SDS and incubated at 65 oC for 10 min, then quenched by Triton X-100. The chromatin was digested by 600 U MboI (NEB, Ipswich, USA) at 37 oC overnight. The next day, enzymes were inactivated by 10% SDS at 65 oC for 30 min, and the restriction fragment overhangs were filled in using biotin-14-dCTP and Klenow at 37 oC for 45 min, and the ligation was conducted in 7.61 ml ligation mix (745 μl 10% Triton X-100, 745 μl 10x ligation buffer (500 mM Tris-HCl pH 7.5, 100 mM MgCl2, 100 mM DTT), 80 μl 10 mg/ml BSA, 80 μl 100 mM ATP and 5.96 ml water) for 4 h at 16°C. The blunt-end Hi-C ligation was sonicated and pull down by streptavidin beads, then purified by ethanol. Hi-C library was prepared for paired ending sequencing using NEBNext® UltraTM DNA Library Prep Kit for Illuina® (NEB). For regular 3C ligation, after enzymes inactivation, 10 μl 1 U/μl T4 DNA ligase was added to incubate at 16°C overnight. The interaction was detected by qPCR using the specific 3C primers (Table 1) followed by DNA purification. Hi-C was conducted in PE-150. An iterative method for Hi-C mapping with initial length 30bp, if it fails to map uniquely, the next round of mapping for additional 20 bp continued, this procedure lasts until the full read length reaches 150bp. The Hi-C reads were mapped to the human reference genome (assembly GRCh38) using bowtie2 (v2.2.9). The reads mapped to the same restriction fragment, the reads less than 500bp and PCR duplicates were all removed. Hi-C contact maps were normalized using ICE.

			Action	DRS
	QCpool_1.raw	Raw
	QCpool_1_150210140013.raw	Raw
	QCpool_2.raw	Raw
	QCpool_3.raw	Raw
	QCpool_4.raw	Raw

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S. cerevisiae proteome response to heat stress - 240 min time course. C4PR_LIV

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