|
| Status |
Public on Mar 14, 2013 |
| Title |
ChIP_Top3-Myc_30C |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
ChIP DNA, Top3-Myc, 30C
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: Hu1747
genotype/variation: WT Top3-myc
temperature: 30C
chip antibody: 9E10 mouse anti-Myc
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 |
Top3-Myc was immunoprecipitated from chromatin extracts prepared from mid-logarithmic phase cells at 30C. 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 9E10 mouse anti-Myc 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, WT, 30C
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: Hu303
genotype/variation: WT
temperature: 30C
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 |
Top3-Myc was immunoprecipitated from chromatin extracts prepared from mid-logarithmic phase cells at 30C. 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 9E10 mouse anti-Myc 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:
WT Top3-myc 30C A.CEL
WT Top3-myc 30C B.CEL
ctrl CEL files:
JW_1 WT 30C.CEL
JW_2_2 WT 30C.CEL
Top3-myc ChIP DNA from cells expressing Top3-Myc from the endogenous promoter at 30C
ChIP_Top3-Myc_30C
|
| 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 |
| GSM1080764_JW_1_WT_30C.CEL.gz |
12.8 Mb |
(ftp)(http) |
CEL |
| GSM1080764_JW_2_2_WT_30C.CEL.gz |
13.4 Mb |
(ftp)(http) |
CEL |
| GSM1080764_Top3myc_A+B_Input_1+2_separate_2004_Fwd_signal.bar.gz |
3.3 Mb |
(ftp)(http) |
BAR |
| GSM1080764_Top3myc_A+B_Input_1+2_separate_2004_Fwd_signal.txt.gz |
3.4 Mb |
(ftp)(http) |
TXT |
| GSM1080764_Top3myc_A+B_Input_1+2_separate_2004_Rev_signal.bar.gz |
3.3 Mb |
(ftp)(http) |
BAR |
| GSM1080764_Top3myc_A+B_Input_1+2_separate_2004_Rev_signal.txt.gz |
3.4 Mb |
(ftp)(http) |
TXT |
| GSM1080764_Top3myc_A+B_Input_1+2_separate_2007_Fwd_signal.bar.gz |
3.0 Mb |
(ftp)(http) |
BAR |
| GSM1080764_Top3myc_A+B_Input_1+2_separate_2007_Fwd_signal.txt.gz |
3.1 Mb |
(ftp)(http) |
TXT |
| GSM1080764_Top3myc_A+B_Input_1+2_separate_2007_Rev_signal.bar.gz |
3.0 Mb |
(ftp)(http) |
BAR |
| GSM1080764_Top3myc_A+B_Input_1+2_separate_2007_Rev_signal.txt.gz |
3.1 Mb |
(ftp)(http) |
TXT |
| GSM1080764_WT_Top3-myc_30C_A.CEL.gz |
14.0 Mb |
(ftp)(http) |
CEL |
| GSM1080764_WT_Top3-myc_30C_B.CEL.gz |
12.6 Mb |
(ftp)(http) |
CEL |
| Processed data provided as supplementary file |
