|
| Status |
Public on Mar 06, 2015 |
| Title |
H3-HA(RITE)_2h_B |
| Sample type |
genomic |
| |
|
| Source name |
H3-HA(RITE), 2h
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: Hu2549
genotype: H3.2-lox-HA-hygR-Lox-T7, cdc25-22 (ts)
chip antibody: HA (abcam ab9110, lot GR98618-1)
sample type: ChIP DNA
treatment: 36°C (5h) + estradiol (2h)
|
| Growth protocol |
Liquid cultures were grown in YES media shaking at 180 rpm at 30 °C (or 25°C for Hu2549).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Immunoprecipitation was done using indicated antibody from chromatin extracts prepared from mid-logarithmic phase cells. 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 |
H3-HA(RITE), 2h
Results reported in:
HA_2_0h_signal.bar
HA_2_0h_B_2004_signal.bar
|
| Data processing |
CEL.files were analysed using Affymetrix Tiling Analysis Software (TAS) v1.1. Two sample comparisons of 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 (WT vs. set9, or 2h vs. 0h) for each probe.
|
| |
|
| Submission date |
Jul 16, 2014 |
| Last update date |
Mar 07, 2015 |
| 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 (2) |
| GSE59461 |
A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depend on histone recycling in transcribed chromatin [ChIP] |
| GSE66866 |
A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depend on histone recycling in transcribed chromatin |
|
