|
| Status |
Public on Jan 29, 2014 |
| Title |
H3K9me2_Hu303 |
| Sample type |
genomic |
| |
|
| Source name |
ChIP DNA, WT, H3K9me2
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: Hu303
genotype: WT
chip antibody: H3K9me2 (07-441, lot DAM1463717, EMD Millipore)
sample type: ChIP DNA
|
| Growth protocol |
Liquid cultures were grown in YES media shaking at 180 rpm at 30 °C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Immunoprecipitation was done using indicated antibody 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 indicated antibody 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 |
ChIP DNA, WT, H3K9me2
Results file: H3K9me2_WT_input.bar
Results file: H3K9me2_Hu2640_WT.bar
Additional results file: H3K9me2_WT_input.txt
Additional results file: H3K9me2_Hu2640_WT.txt
|
| Data processing |
CEL.files were analysed using Affymetrix Tiling Analysis Software (TAS) v1.1. Two sample comparisons of immunoprecipitated vs. input samples or immunoprecipitated (strain 1) vs. immunoprecipitated (strain 2), quantile normalization (separate) and linear scaling to 100 was applied. Probe signals were generated using a bandwidth of 100 and assigned to S. pombe genome coordinates (Sanger 2004) and reported in BAR files.
Results files in BAR formats were generated using Affymetrix Tiling Analysis Software (TAS) v1.1. and contain log2 values for relative enrichment (ChIP vs. Input, or ChIP vs. ChIP) for each probe.
Additional results files in txt formats were generated using Affymetrix Tiling Analysis Software (TAS) v1.1. and contain log2 values for relative enrichment (ChIP vs. Input, or ChIP vs. ChIP) for each probe.
|
| |
|
| Submission date |
Aug 14, 2013 |
| Last update date |
Jan 30, 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) |
| GSE49874 |
Centromeric H2B monoubiquitination promotes noncoding transcription and chromatin integrity |
|
