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Sample GSM1080766 Query DataSets for GSM1080766
Status Public on Mar 14, 2013
Title ChIP_Cnp1_top3-105_36C
Sample type genomic
 
Channel 1
Source name ChIP DNA, Cnp1, top3-105, 36C
Organism Schizosaccharomyces pombe
Characteristics strain: Hu2027
genotype/variation: top3-105
temperature: 36C
chip antibody: anti-Cnp1 serum
sample type: ChIP DNA
Growth protocol Liquid cultures were grown in YES media shaking at 180 rpm. The temperature sensitive top3-105 mutant and wild type controls were grown at the permissive temperature (25 °C) over night and then for 8 hours at the nonpermissive temperature (36°C) before harvesting. All other strains were grown at 30 °C.
Extracted molecule genomic DNA
Extraction protocol Cnp1 was immunoprecipitated from chromatin extracts prepared from mid-logarithmic phase cells after 8 hours at 36C. Briefly, cells were fixed in 1% formaldehyde for 30 minutes, quenched with 125 mM glycine, and washed twice in PBS. Cells were resuspended in lysis buffer (0.1% SDS, 50 mM Hepes-KOH, 1 % Triton X-100, 0.1 % sodium deoxucholate, 1 mM EDTA and 150 mM NaCl) and lysed with glass beads in a FastPrep homogenizer. The lysate was sonicated and soluble chromatin fragments with an average size of 600 bp were collected. Chromatin was immunoprecipitated with anti-Cnp1 serum for 2 hours and incubated with protein A agarose beads for 1 hour. The beads were washed, chromatin eluted at with TES at 65C and crosslinks reversed at 65C over night, together with input samples. Proteins were digested with proteinase K and immunoprecipitated DNA recovered using QIAquick PCR Purification kit.
Label biotin
Label protocol A specific sequence tag was attached to immunoprecipitated DNA fragments by PCR using sequenase and random primers (5'-GTTTCCCAGTCACGATCNNNNNNNNN-3'). This DNA was amplified using Taq polymerase and a primer recognizing the specific sequence tag (5'-GTTTCCCAGTCACGATC-3') in the precence of 5 mM dUTP. PCR products were purified using Qiaquick PCR purification kit. DNA was fragmented by UDG and APE nuclease treatment and labelled with biotin by Affymetrix core facility at Novum (http://apt.bea.ki.se) according to standard Affymetrix protocols. Immunoprecipitation Assay Protocol
 
Channel 2
Source name ChIP input DNA, top3-105, 36C
Organism Schizosaccharomyces pombe
Characteristics strain: Hu2027
genotype/variation: top3-105
temperature: 36C
sample type: input DNA
Growth protocol Liquid cultures were grown in YES media shaking at 180 rpm. The temperature sensitive top3-105 mutant and wild type controls were grown at the permissive temperature (25 °C) over night and then for 8 hours at the nonpermissive temperature (36°C) before harvesting. All other strains were grown at 30 °C.
Extracted molecule genomic DNA
Extraction protocol Cnp1 was immunoprecipitated from chromatin extracts prepared from mid-logarithmic phase cells after 8 hours at 36C. Briefly, cells were fixed in 1% formaldehyde for 30 minutes, quenched with 125 mM glycine, and washed twice in PBS. Cells were resuspended in lysis buffer (0.1% SDS, 50 mM Hepes-KOH, 1 % Triton X-100, 0.1 % sodium deoxucholate, 1 mM EDTA and 150 mM NaCl) and lysed with glass beads in a FastPrep homogenizer. The lysate was sonicated and soluble chromatin fragments with an average size of 600 bp were collected. Chromatin was immunoprecipitated with anti-Cnp1 serum for 2 hours and incubated with protein A agarose beads for 1 hour. The beads were washed, chromatin eluted at with TES at 65C and crosslinks reversed at 65C over night, together with input samples. Proteins were digested with proteinase K and immunoprecipitated DNA recovered using QIAquick PCR Purification kit.
Label biotin
Label protocol A specific sequence tag was attached to immunoprecipitated DNA fragments by PCR using sequenase and random primers (5'-GTTTCCCAGTCACGATCNNNNNNNNN-3'). This DNA was amplified using Taq polymerase and a primer recognizing the specific sequence tag (5'-GTTTCCCAGTCACGATC-3') in the precence of 5 mM dUTP. PCR products were purified using Qiaquick PCR purification kit. DNA was fragmented by UDG and APE nuclease treatment and labelled with biotin by Affymetrix core facility at Novum (http://apt.bea.ki.se) according to standard Affymetrix protocols. Immunoprecipitation Assay Protocol
 
 
Hybridization protocol Hybridised to Affymetrix GeneChip S. pombe Tiling 1.0FR Affymetrix core facility at Novum (http://apt.bea.ki.se) according to standard Affymetrix protocols.
Scan protocol Scanned at the Affymetrix core facility at Novum (http://apt.bea.ki.se) according to standard Affymetrix protocols.
Description test CEL files:
Cnp1 Top3-105 8h 36C A.CEL
Cnp1 Top3-105 8h 36C B.CEL
ctrl CEL files:
Input Top3-105 8h 36C B.CEL
Cnp1 ChIP DNA from top5-105 cells after 8 hours at 36C
ChIP_Cnp1_top3-105_36C
Data processing CEL.files were analysed using Affymetrix Tiling Analysis Software (TAS) v1.1. Two sample comparisons of immunoprecipitated vs. input samples, quantile normalization (separate) and linear scaling to 100 was applied. Linear probe signals were generated using a bandwidth of 100 and assigned to S. pombe genome coordinates (Sanger 2007 and Sanger 2004) and reported in BAR files (Fwd and Rev strands).
All result files are also provided in .txt format: 2007 Transcription Hu303 A+B (one sample PM MM) Fwd_linear signal.txt, 2007 Transcription Hu303 A+B (one sample PM MM) Rev_linear signal.txt, 2007 Transcription Hu2027 A+B (one sample PM MM) Fwd_linear signal.txt, 2007 Transcription Hu2027 A+B (one sample PM MM) Rev_linear signal.txt, Top3myc_A+B_Input_1+2 (separate) 2004 Fwd_signal.txt, Top3myc_A+B_Input_1+2 (separate) 2004 Rev_signal.txt, Top3myc_A+B_Input_1+2 (separate) 2007 Fwd_signal.txt, Top3myc_A+B_Input_1+2 (separate) 2007 Rev_signal.txt, Cnp1 WT 36C_A+B_Input WT 36C_B (separate) 2004 Fwd_signal.txt, Cnp1 WT 36C_A+B_Input WT 36C_B (separate) 2004 Rev_signal.txt, Cnp1 WT 36C_A+B_Input WT 36C_B (separate) 2007 Fwd_signal.txt, Cnp1 WT 36C_A+B_Input WT 36C_B (separate) 2007 Rev_signal.txt, Cnp1 Top3-105 36C_A+B_Input Top3-105 36C_B (separate) 2004 Fwd_signal.txt, Cnp1 Top3-105 36C_A+B_Input Top3-105 36C_B (separate) 2004 Rev_signal.txt, Cnp1 Top3-105 36C_A+B_Input Top3-105 36C_B (separate) 2007 Fwd_signal.txt, Cnp1 Top3-105 36C_A+B_Input Top3-105 36C_B (separate) 2007 Rev_signal.txt
Results file in .BAR and .txt formats were generated using Affymetrix Tiling Analysis Software (TAS) v1.1. and contains linear values for transcription in arbitrary units or relative enrichment (ChIP vs. input) for each probe.
 
Submission date Feb 08, 2013
Last update date Mar 14, 2013
Contact name Karl Ekwall
E-mail(s) karl.ekwall@ki.se
Phone +46 8 6089133
Organization name Karolinska Inst
Street address Alfred Nobels Alle 7
City Stockholm
ZIP/Postal code S-141 89
Country Sweden
 
Platform ID GPL7715
Series (1)
GSE44206 DNA topoisomerase III localizes to centromeres and affects centromeric CENP-A levels in fission yeast

Supplementary file Size Download File type/resource
GSM1080766_Cnp1_Top3-105_36C_A+B_Input_Top3-105_36C_B_separate_2004_Fwd_signal.bar.gz 3.3 Mb (ftp)(http) BAR
GSM1080766_Cnp1_Top3-105_36C_A+B_Input_Top3-105_36C_B_separate_2004_Fwd_signal.txt.gz 3.3 Mb (ftp)(http) TXT
GSM1080766_Cnp1_Top3-105_36C_A+B_Input_Top3-105_36C_B_separate_2004_Rev_signal.bar.gz 3.3 Mb (ftp)(http) BAR
GSM1080766_Cnp1_Top3-105_36C_A+B_Input_Top3-105_36C_B_separate_2004_Rev_signal.txt.gz 3.3 Mb (ftp)(http) TXT
GSM1080766_Cnp1_Top3-105_36C_A+B_Input_Top3-105_36C_B_separate_2007_Fwd_signal.bar.gz 3.0 Mb (ftp)(http) BAR
GSM1080766_Cnp1_Top3-105_36C_A+B_Input_Top3-105_36C_B_separate_2007_Fwd_signal.txt.gz 3.0 Mb (ftp)(http) TXT
GSM1080766_Cnp1_Top3-105_36C_A+B_Input_Top3-105_36C_B_separate_2007_Rev_signal.bar.gz 3.0 Mb (ftp)(http) BAR
GSM1080766_Cnp1_Top3-105_36C_A+B_Input_Top3-105_36C_B_separate_2007_Rev_signal.txt.gz 3.0 Mb (ftp)(http) TXT
GSM1080766_Cnp1_Top3-105_8h_36C_A.CEL.gz 11.0 Mb (ftp)(http) CEL
GSM1080766_Cnp1_Top3-105_8h_36C_B.CEL.gz 9.2 Mb (ftp)(http) CEL
GSM1080766_Input_Top3-105_8h_36C_B.CEL.gz 11.2 Mb (ftp)(http) CEL
Processed data provided as supplementary file

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