|
| Status |
Public on Aug 22, 2013 |
| Title |
ChIP_Endog. Cnp1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
ChIP DNA, Cnp1, ENDOGENOUS
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: 1645
chip antibody: Cnp1
|
| Growth protocol |
Cells expressing additional CENP-ACnp1 from integrated pREP81-cnp1+ (nmt81-CENP-ACnp1), pREP41-cnp1+ (nmt41-CENP-ACnp1) or cells with integrated empty vector were initially grown in rich medium which contains thiamine to repress the expression of additional CENP-ACnp1. Cells were grown in PMG liquid medium (with thiamine) at 25ºC and shifted to 36ºC in PMG lacking thiamine for 24 h to allow CENP-ACnp1 expression before ChIP analysis.
|
| 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, WT
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: 1645
chip antibody: none
|
| Growth protocol |
Cells expressing additional CENP-ACnp1 from integrated pREP81-cnp1+ (nmt81-CENP-ACnp1), pREP41-cnp1+ (nmt41-CENP-ACnp1) or cells with integrated empty vector were initially grown in rich medium which contains thiamine to repress the expression of additional CENP-ACnp1. Cells were grown in PMG liquid medium (with thiamine) at 25ºC and shifted to 36ºC in PMG lacking thiamine for 24 h to allow CENP-ACnp1 expression before ChIP analysis.
|
| 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 |
Cnp1 ChIP DNA from WT cells after 8 hours at 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: Input_2.txt, Input_1 .txt, WT_oe_cnp1_4.txt, WT_oe_Cnp1_3.txt, WT_oe_Cnp1_2.txt, WT_oe_Cnp1_1.txt, WT_Cnp1_2.txt, WT_Cnp1_1.txt
Results file in .BAR and .txt formats were generates using Affymetrix Tiling Analysis Software (TAS) v1.1. and contains linear values for relative enrichment (ChIP vs. input) for each probe.
|
| |
|
| Submission date |
Apr 26, 2013 |
| Last update date |
Jan 06, 2014 |
| 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) |
| GSE46427 |
Telomeric repeats facilitate CENP-A incorporation via telomere binding proteins |
|
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1130059_1._Cnp1_WT_signal.bar.gz |
5.7 Mb |
(ftp)(http) |
BAR |
| GSM1130059_1._Cnp1_WT_signal.bar.txt.gz |
6.1 Mb |
(ftp)(http) |
TXT |
| GSM1130059_Input_1_.CEL.gz |
12.8 Mb |
(ftp)(http) |
CEL |
| GSM1130059_Input_1_.txt.gz |
18.7 Mb |
(ftp)(http) |
TXT |
| GSM1130059_Input_2.CEL.gz |
13.4 Mb |
(ftp)(http) |
CEL |
| GSM1130059_Input_2.txt.gz |
19.1 Mb |
(ftp)(http) |
TXT |
| GSM1130059_WT_Cnp1_1.CEL.gz |
12.4 Mb |
(ftp)(http) |
CEL |
| GSM1130059_WT_Cnp1_1.txt.gz |
18.2 Mb |
(ftp)(http) |
TXT |
| GSM1130059_WT_Cnp1_2.CEL.gz |
13.3 Mb |
(ftp)(http) |
CEL |
| GSM1130059_WT_Cnp1_2.txt.gz |
19.0 Mb |
(ftp)(http) |
TXT |
| Processed data provided as supplementary file |
