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Sample GSM1263360

Query DataSets for GSM1263360

Status

Public on Aug 31, 2014

Title

H3K9ac_time alignment_ChIP-seq_t6

Sample type

SRA

Source name

YMC cycling cells

Organism

Saccharomyces cerevisiae

Characteristics

strain: BY5765: CEN.PK MATalpha
time: t6
chip antibody: H3K9ac [06-942 (Millipore)]

Extracted molecule

genomic DNA

Extraction protocol

~50 OD cycling cells per time point were collected for ChIP 3 µg primary antibody was used per ChIP experiment. Briefly, cells were first fixed in 1% formaldehyde at 25℃ for 15 min and quenched in 125mM glycine at 25℃ for 10 min. Cells were pelleted and washed twice with buffer containing 100 mM NaCl, 10 mM Tris-Cl pH 8.0, 1 mM EDTA, 1 mM PMSF, 1 mM benzamidine•HCl before freezing. The frozen pellet was resuspended in 0.45 ml ChIP lysis buffer (50 mM HEPES•KOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% deoxycholate (DOC), 0.1% SDS, 1 mM PMSF, 10 µM leupeptin, 5 µM pepstatin A, Roche protease inhibitor cocktail) and lysed by bead beating. Lysate from 50 OD cells were split into two tubes each containing 280 µl lysate and sonicated for 16 cycles (30 sec on, 1 min off, high output) using a Bioruptor (Diagenode, Denville, NJ or Tosho Denki, Japan). The supernatant of the sonicated lysate was pre-cleared. 50 µl lysate was saved as input. For ChIP with histone antibodies, 50 µl whole cell extract (WCE) was diluted 1:10 and used for each ChIP. After incubation overnight, 50 µl protein G magnetic beads (Invitrogen, Grand Island, NY, 10003D) resuspended in ChIP lysis buffer was added and incubated for 1.5 h at 4 ℃. Beads were washed twice with ChIP lysis buffer, twice with DOC buffer (10 mM Tris•Cl pH 8.0, 0.25 M LiCl, 0.5% deoxycholate, 0.5% NP-40, 1 mM EDTA) and twice with TE. 125 µl of TES buffer (TE pH8.0 with 1% SDS, 150 mM NaCl, and 5 mM dithiothreitol) was added to resuspend the beads. Supernatant was collected after incubation at 65°C for 10 min. A second round of elution was performed and the eluates were combined. Reverse crosslinking was performed by incubation for 6 h at 65°C. An equal volume of TE containing 1.25 mg/ml proteinase K and 0.4 mg/ml glycogen was added to the samples after reverse crosslinking and samples were incubated for 2 h at 37°C. Samples were extracted twice with an equal volume of phenol and once with 25:1 chloroform:isoamyl alcohol. DNA was precipitated in 0.1 volume 3.0 M sodium acetate (PH 5.3) and 2.5 volume of 100% ice-cold ethanol at -20℃ overnight. Pellets were washed once with cold 70% ethanol and resuspended in 20 µl TE.
Library construction and sequencing were performed following the Illumina protocol. Briefly, DNA was end repaired and A-tailed. Barcoded adaptors were ligated and DNA was run in 2% agarose gel. DNA fragments from 150 bp to 300 bp long were excised from gel and used for PCR. PCR products were gel-extracted again and quantified on an Agilent Bioanalyzer.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Description

Same time points as RNA-Seq. Used for time alignment between RNA-Seq and histone modification ChIP-Seq experiments.

Data processing

Sequence reads were mapped to the Saccharomyces.cerevisiae (sacCer2) using bowtie. In order to adjust sequencing depth difference, signals at each window were normalized by total number of mappable reads across 16 time points.
Genome_build: sacCer2

Submission date

Nov 13, 2013

Last update date

May 15, 2019

Contact name

Zheng Kuang

Organization name

UT Southwestern medical center

Department

Immunology

Lab

Lora Hooper