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Sample GSM696101 Query DataSets for GSM696101
Status Public on Jul 25, 2012
Title SET2 deletion AcH4 IP Replicate 2 (MDY510)
Sample type genomic
 
Channel 1
Source name SET2 deletion AcH4 IP
Organism Saccharomyces cerevisiae
Characteristics strain: Exchange strain MDY510
antibody: AcH4
Growth protocol The exchange strains were grown in YP + 2% raffinose at 25°C to an OD of 0.5. a-factor was added to a final concentration of 1 mM and the cells were grown in raffinose containing medium for an additional 4 hrs. After the a-factor arrest, cells were collected washed in 1x PBS with protease inhibitors, resuspended and grown in YP + 2% galactose with 1 mM a-factor at 25°C for 45 min. Cells were cross-linked with 1% formaldehyde at room temperature for 15 min. with continuous shaking. The reaction was quenched by adding glycine to a final concentration of 125mM. Cells were collected by centrifugation and processed for the ChIP assay
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 Flag, Myc and acetylated H4. 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. Crosslinkss 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 37°C, followed by phenol:chloroform extraction and ethanol precipitation. The CIP-treated template was incubated with 20 U TdT (NEB) for 20 minutes at 37°C 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 37°C, followed by phenol:chloroform extraction and ethanol precipitation. In vitro transcription was performed at 37°C 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). 2.5-4µgg 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 strain: Exchange strain MDY510
antibody: --
Growth protocol The exchange strains were grown in YP + 2% raffinose at 25°C to an OD of 0.5. a-factor was added to a final concentration of 1 mM and the cells were grown in raffinose containing medium for an additional 4 hrs. After the a-factor arrest, cells were collected washed in 1x PBS with protease inhibitors, resuspended and grown in YP + 2% galactose with 1 mM a-factor at 25°C for 45 min. Cells were cross-linked with 1% formaldehyde at room temperature for 15 min. with continuous shaking. The reaction was quenched by adding glycine to a final concentration of 125mM. Cells were collected by centrifugation and processed for the ChIP assay
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 Flag, Myc and acetylated H4. 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. Crosslinkss 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 37°C, followed by phenol:chloroform extraction and ethanol precipitation. The CIP-treated template was incubated with 20 U TdT (NEB) for 20 minutes at 37°C 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 37°C, followed by phenol:chloroform extraction and ethanol precipitation. In vitro transcription was performed at 37°C 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). 2.5-4µgg 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 65°C. 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 SET2 deletion exchange strain.
Data processing Data processed in GeneSpring
 
Submission date Mar 22, 2011
Last update date Jul 25, 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)
GSE28096 Histone exchange and histone H4 acetylation in wild-type and SET2 deletion yeast strains
GSE28099 Co-transcriptional histone acetylation is a consequence of histone exchange

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

Data table
ID_REF VALUE
ScCGHBrightCorner -1.1116912
DarkCorner 2.3398135
A_75_P01725145 0.001925436
A_75_P01760060 -0.69560325
A_75_P01353403 -1.6358546
A_75_P01304758 1.3031511
A_75_P01688477 1.1618091
A_75_P02025438 -1.7373495
A_75_P01312283 0.88630486
A_75_P01431373 1.1953887
A_75_P01784551 -0.39140698
A_75_P01297739 -1.0574236
A_75_P01072850 -0.6151218
A_75_P01472698 0.3664979
A_75_P02176820 -0.2728095
A_75_P01825612 0.8009138
A_75_P01645706 0.40497583
A_75_P01933516 -0.32985207
A_75_P02089750 -0.40790755
A_75_P01674052 0.13475515

Total number of rows: 43502

Table truncated, full table size 1100 Kbytes.




Supplementary file Size Download File type/resource
GSM696101_mdyset2_AcH4_2.txt.gz 4.7 Mb (ftp)(http) TXT
Processed data included within Sample table

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