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Sample GSM909268 Query DataSets for GSM909268
Status Public on Jul 25, 2012
Title H3K36A H3 IP Replicate 2
Sample type genomic
 
Channel 1
Source name IP DNA H3 antibody
Organism Saccharomyces cerevisiae
Characteristics strain: YBL574
chip antibody: anti H3
genotype: H3K36A
Extracted molecule genomic DNA
Extraction protocol Lysates were prepared by bead beating in FA lysis buffer (50mM HEPES pH7.5, 1mM EDTA, 1% Triton X-100, 0.1%sodium deoxycholate and 150mM NaCl). The ChIP lysates were used to immunoprecipitate histone H3K56 acetylated nucleosomes. The immune complexes were pulled down with 50 ml of Protein G Dynabeads (Invitrogen) per IP and washed with FA lysis buffer, FA lysis buffer with 1M NaCl, FA lysis buffer with 0.5M NaCl, TEL buffer (0.25M LiCl, 1%NP-40, 1% sodium deoxycholate, 1mM EDTA, 10mM TrisHCL, pH8.1), and twice with TE. The Immune complexes were eluted with Elution buffer (1%SDS in TE with250mM NaCl). The eluates were RNase treated and Proteinase K was added to remove the bound proteins. Crosslinks were reversed by incubating at 65 C overnight. Samples werte phenol:chloroform treated and ethanol precipitated. Input samples were treated the same way from the RNase step.
Label cy5
Label protocol Briefly, up to 50 ng of IP or input DNA was treated with 2.5 U CIP enzyme (NEB) for 1 hour at 37C, followed by phenol:chloroform extraction and ethanol precipitation. The CIP-treated template was incubated with 20 U TdT (NEB) for 20 minutes at 37C for T-tailing the ends, and the product isolated with the MinElute Reaction Cleanup Kit (Qiagen). An anchored T7 promoter-(dA)18 oligo was added by annealing and filling with 5 U Exo- Klenow (NEB) for 4 hours at 37C, followed by phenol:chloroform extraction and ethanol precipitation. In vitro transcription was performed at 37C overnight using the Ampliscribe T7 transcription kit (Epicentre Biotechnologies). RNAs purified using the RNeasy Mini Kit (Qiagen) and quantified on a Nanodrop 2000 Spectrophotometer (Thermo Scientific). For the second round amplification, 100–150 ng of RNA was reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and purified with the MinElute Reaction Cleanup Kit (Qiagen). The T7 promoter primer was added to the product through a Klenow filling reaction as described above, followed by in vitro transcription with amino allyl-UTP (Ambion) instead of UTP. Final reaction cleanup was performed with the RNeasy Mini Kit (Qiagen). For the labeling reactions, 8 ug of amino allyl-incorporated RNA in a 5 uL volume of 0.1 M Carbonate Buffer, pH 8.7, was mixed with 5 uL (0.01 nmol) Cy3 or Cy5 dye (GE Healthcare) in DMSO (Sigma) and incubated at room temperature for 2 hours. Reactions were quenched with 5 uL 4 M hydroxylamine at room temperature for 15 minutes, purified by RNeasy MinElute Cleanup Kit (Qiagen), and the dye incorporation measured using the Nanodrop 2000 (Thermo Scientific). 2.5-4 ug of input and IP were combined and fragmented (Fragmentation Reagent Kit, Ambion) according to manufacturer’s instructions.
 
Channel 2
Source name Input DNA
Organism Saccharomyces cerevisiae
Characteristics genotype: H3K36A
strain: YBL574
chip antibody: none
Extracted molecule genomic DNA
Extraction protocol Lysates were prepared by bead beating in FA lysis buffer (50mM HEPES pH7.5, 1mM EDTA, 1% Triton X-100, 0.1%sodium deoxycholate and 150mM NaCl). The ChIP lysates were used to immunoprecipitate histone H3K56 acetylated nucleosomes. The immune complexes were pulled down with 50 ml of Protein G Dynabeads (Invitrogen) per IP and washed with FA lysis buffer, FA lysis buffer with 1M NaCl, FA lysis buffer with 0.5M NaCl, TEL buffer (0.25M LiCl, 1%NP-40, 1% sodium deoxycholate, 1mM EDTA, 10mM TrisHCL, pH8.1), and twice with TE. The Immune complexes were eluted with Elution buffer (1%SDS in TE with250mM NaCl). The eluates were RNase treated and Proteinase K was added to remove the bound proteins. Crosslinks were reversed by incubating at 65 C overnight. Samples werte phenol:chloroform treated and ethanol precipitated. Input samples were treated the same way from the RNase step.
Label cy3
Label protocol Briefly, up to 50 ng of IP or input DNA was treated with 2.5 U CIP enzyme (NEB) for 1 hour at 37C, followed by phenol:chloroform extraction and ethanol precipitation. The CIP-treated template was incubated with 20 U TdT (NEB) for 20 minutes at 37C for T-tailing the ends, and the product isolated with the MinElute Reaction Cleanup Kit (Qiagen). An anchored T7 promoter-(dA)18 oligo was added by annealing and filling with 5 U Exo- Klenow (NEB) for 4 hours at 37C, followed by phenol:chloroform extraction and ethanol precipitation. In vitro transcription was performed at 37C overnight using the Ampliscribe T7 transcription kit (Epicentre Biotechnologies). RNAs purified using the RNeasy Mini Kit (Qiagen) and quantified on a Nanodrop 2000 Spectrophotometer (Thermo Scientific). For the second round amplification, 100–150 ng of RNA was reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and purified with the MinElute Reaction Cleanup Kit (Qiagen). The T7 promoter primer was added to the product through a Klenow filling reaction as described above, followed by in vitro transcription with amino allyl-UTP (Ambion) instead of UTP. Final reaction cleanup was performed with the RNeasy Mini Kit (Qiagen). For the labeling reactions, 8 ug of amino allyl-incorporated RNA in a 5 uL volume of 0.1 M Carbonate Buffer, pH 8.7, was mixed with 5 uL (0.01 nmol) Cy3 or Cy5 dye (GE Healthcare) in DMSO (Sigma) and incubated at room temperature for 2 hours. Reactions were quenched with 5 uL 4 M hydroxylamine at room temperature for 15 minutes, purified by RNeasy MinElute Cleanup Kit (Qiagen), and the dye incorporation measured using the Nanodrop 2000 (Thermo Scientific). 2.5-4 ug of input and IP were combined and fragmented (Fragmentation Reagent Kit, Ambion) according to manufacturer’s instructions.
 
 
Hybridization protocol The hybridization mixture (Oligo CGH/Chip-on-chip hybridization kit, Agilent) was prepared according to manufacturer’s instructions with the addition of 20 ug of T7 blocking oligo (ttt ttt ttt ttt tt cac gcg tct ccc) , and hybridized overnight at 65C. Microarrays were washed using the Oligo aCCH/Chip-on-chip Wash buffers (Agilent) according to manufacturer’s instructions.
Scan protocol Scanned on Agilent Technologies Scanner G2505C US45102919
Images were quantified using Agilent Feature Extraction Software (version 10.5.1.1).
Description Biological Replicate 2 of 3 ChIP enrichment of AcH4 IP over Input in the WT BY4741 strain
Data processing Data processed on R. Median normalization carried out.
 
Submission date Apr 04, 2012
Last update date Jul 26, 2012
Contact name Swaminathan Venkatesh
E-mail(s) [email protected]
Phone 816-926-4312
Fax 816-926-4866
Organization name Stowers Institute for Medical Research
Lab Workman Lab
Street address 1000 E 50th Street
City Kansas City
State/province MO
ZIP/Postal code 64112
Country USA
 
Platform ID GPL10930
Series (2)
GSE28099 Co-transcriptional histone acetylation is a consequence of histone exchange
GSE37039 Histone H3 K56 acetylation in wild-type histone shuffle strain (YBL574)

Data table header descriptions
ID_REF
VALUE Normalized Log2 ratio of IP over Input

Data table
ID_REF VALUE
A_75_P01725145 -0.495020865
A_75_P01760060 -1.695189556
A_75_P01353403 -0.527787946
A_75_P01304758 0.63801344
A_75_P01688477 0.685995825
A_75_P02025438 0.79810333
A_75_P01312283 0.315791467
A_75_P01431373 0.073172481
A_75_P01784551 0.00598485
A_75_P01297739 -0.332323173
A_75_P01072850 0.036907922
A_75_P01472698 0.316821676
A_75_P02176820 -0.160219014
A_75_P01825612 -0.731562019
A_75_P01645706 -0.233876446
A_75_P01933516 -0.265318638
A_75_P02089750 0.296139262
A_75_P01674052 -0.366735421
A_75_P02017800 0.163833091
A_75_P01376352 0.419206976

Total number of rows: 41775

Table truncated, full table size 1117 Kbytes.




Supplementary file Size Download File type/resource
GSM909268_YBLK36A_H3_2.txt.gz 12.8 Mb (ftp)(http) TXT
Processed data included within Sample table

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