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Sample GSM936787

Query DataSets for GSM936787

Status

Public on Jul 15, 2012

Title

Rpb3IP_Dhog1_KCl

Sample type

SRA

Source name

S. cerevisiae liquid culture in logarhithmic growth

Organism

Saccharomyces cerevisiae

Characteristics

target of ip: Rpb3 (untagged)
chip antibody: 1Y26 (neoclone, catalog# WP012, lot# A5081456)
stress treatment: YEPD + 0.4 M KCl for 5 minutes
genetic background: Dhog1

Treatment protocol

For stress treatment, YEPD supplemented with KCl was added to ~120 OD units of culture, bringing the final concentration of KCl to 0.4 M; for mock treated cells, the same volume of YEPD was added to cultures. After five minutes in stress, samples were crosslinked with 1% formaldehyde at room temperature for 15 minutes. Crosslinking was quenched with 125 mM glycine for five minutes, and then samples were harvested by centrifugation, washed twice in cold PBS (137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic, pH 7.4) and then snap-frozen in liquid nitrogen.

Growth protocol

Cells were diluted from an overnight culture to an OD600 of 0.1. Cells were grown at 30° C in YEPD, with shaking, to OD600 0.6. Cultures were split for stress treatment and mock treatment.

Extracted molecule

genomic DNA

Extraction protocol

Samples were resuspended in 1 mL lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton-X 100, 0.1% Na Deoxycholate) in the presence of protease inhibitors (Roche; Complete) and mechanically lysed by bead beading. Lysates were sonicated (9 cycles of 15 seconds each, power 2 on Misonix 3000) to solubilize chromatin and then clarified by centrifugation. Ten percent of the clarified lysate was reserved to serve as an input control. Clarified lysates (~10 mg for ChIP-seq, ~ 2.5 mg for ChIP qPCR) were incubated with 12CA5 anti-HA antibody or 1Y26 anti Rpb3 antibody, for 2 hours at 4° C before addition of Protein G Dynabeads (Invitrogen). After incubation for 4 hours up to overnight, beads were washed twice with lysis buffer, once with high salt buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton-X 100, 0.1% Na Deoxycholate), once with lysis buffer and once with TE (10 mM Tris, 1 mM EDTA, pH 8). All washes were performed at room temperature for five minutes each, on an end-over-end mixer. Samples were eluted from beads in TE plus 0.67% SDS at 65° C for 30 minutes. Supernatants were removed from beads and incubated overnight at 65° C to break crosslinks. DNA was isolated by digestion of RNA with RNAse A for 2 hours at 37° C, protein digestion with Proteinase K for 2 hours at 55° C, and purified by phenol chloroform extraction and precipitation with ethanol and NaCl. DNA pellets were stored in TE.
For ChIP-seq, ~ 10 ng IP material was used to generate each library, following the Illumina protocol for their paired end DNA sample prep kit (v1). After addition of adaptors, DNA in a size range of 175-300 base pairs was isolated by gel electrophoresis for amplification. Size ranges of prepared libraries were measured on an Agilent Biolanalyzer (average size 225 base pairs) before sequencing on an Illumina Genome Analyzer II (performed by Christian Daly at the FAS Center for Systems Biology Core Facility). Thirty-six base reads were obtained and aligned to the Saccharomyces cerevisiae genome using ELAND (Jianwen Zhang performed the alignments). For Hog1 and transcription factor ChIP-seq samples, five million to ten million reads were obtained. Uniquely alignable sequence tags were mapped to the genome and extended by the average length of the library (minus the adaptor length) in MATLAB.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer II

Description

processed data file: ORFcounts.txt

Data processing

base calling with Illumina pipeline
alignment with Bowtie (Langmead et al, 2009) to June 2008 assembly of S. cerevisiae genome
count reads in Matlab, screen out spikes that occur in only one strand (where reads one strand are >10x the other strand)
Genome_build: SGD/sacCer2 assembly (June 2008) from UCSC browser
Supplementary_files_format_and_content: .txt tables with sequencing reads summed within each ORF, promoter region for RNA Pol II and Hog1, and lists of binding peaks for the transcription factors Sko1 and Hot1
The .txt tables are linked as supplementary files on the Series record.

Submission date

May 24, 2012

Last update date

May 15, 2019

Contact name

Kristen E Cook

E-mail(s)

kcook@mcb.harvard.edu

Organization name

Harvard University

Department

Molecular and Cellular Biology