|
| Status |
Public on Dec 20, 2016 |
| Title |
Input_2 |
| Sample type |
genomic |
| |
|
| Source name |
Arabidopsis WT, none
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: aerial parts of plant
ecotype: Col-0
treatment: sonicated chromatin
antibody: none
age: 3-weeks old
|
| Treatment protocol |
Plant tissue was crosslinked in 1% formaldehyde in 1xPBS buffer under vaccume (2x 10 min.). After incubation in 0.125 M glycine for 5 min, plant tissues were washed twice with 1× PBS. Nuclear extracts were prepared as described by Gendrel et al. (2005) and sonicated using Bioruptor sonicator (Diagenode). The extracts were then centrifuged for 5 minutes, 4°C at 10,000 × g and the supernatants were used for immunoprecipitation.
|
| Growth protocol |
Chromatin was isolated from 3-week-old seedlings of wild-type Arabidopsis (Col-0 ecotype) grown in soil under long-day conditions. Aerial parts of plants were harvested and rinsed in water.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Samples were diluted 10-fold with ChIP Dilution Buffer (1.1 % Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, protease inhibitors (Roche). For immunoprecipitation, Anti-BRM antibody [Archacki et al., 2009) was bound to Dynabeads Protein A (Invitrogen) and incubated overnight with aliquotes of diluted chromatin (100 µg of DNA). Beads were washed with Low Salt, High Salt and LiCl, and TE buffer as described (Gendrel et al., 2005). Immune complexes were eluted by adding 500 µl of Elution Buffer (1% SDS, 0.1 M NaHCO3) to the pelleted beads. Cross-links were reversed by addition of 40 ul 2.5 M NaCl and incubation at 65°C overnight. DNA was purified with a PCR purification kit (Qiagen) and subjected to two rounds of amplification using a WGA2 GenomePlex Complete whole genome amplification kit (Sigma-Aldrich), according to the manufacturer’s protocol.
|
| Label |
biotin
|
| Label protocol |
Four µg of DNA was used for fragmentation and labeling according to Affymetrix chromatin immunoprecipitation assay protocol (P/N 702238 Rev.4) using a GeneChip WT Terminal Labeling Kit (Affymetrix).
|
| |
|
| Hybridization protocol |
Labeled DNA was hybridized to Arabidopsis Agronomics microarray (Rehrauer et al., 2010) using a GeneChip Hybridization Wash and Stain Kit according to the manufacturer’s recommendations (Affymetrix).
|
| Scan protocol |
Microarrays were scanned using the Affymetrix GeneChip Scanner 3000. The signals were recorded and processed using Affymetrix® GeneChip® Command Console® Software (AGCC).
|
| Description |
fraction: nuclear DNA
sonicated chromatin
|
| Data processing |
ChIP-chip microarray probe signals were extracted using Affymetrix APT-cel software.
The resulting bed files were generated by the Affymetrix APT-cel software. SGR files contain the normalized probe intensities. Signals for both strands were merged for each replicate and then transformed to TAIR10 coordinates. The signal was normalized separately for each of the two biological replicates and the average of the replicates for each probe was used. Log2 (IP/Input) was computed for every probe and the significant regions were then called by finding regions of length > 40 probes with enrichment > 1.5 (log2 enrichment >0.58), excluding at most 3 faulty probes. The BED file on the series record contains the significantly enriched regions.
|
| |
|
| Submission date |
Aug 03, 2015 |
| Last update date |
Dec 20, 2016 |
| Contact name |
Rafal Archacki |
| E-mail(s) |
rafa@ibb.waw.pl
|
| Organization name |
University of Warsaw
|
| Lab |
Laboratory of Systems Biology
|
| Street address |
Pawinskiego 5A
|
| City |
Warsaw |
| ZIP/Postal code |
02-106 |
| Country |
Poland |
| |
|
| Platform ID |
GPL20763 |
| Series (2) |
| GSE71656 |
Genome-wide profiling of BRM chromatin remodeler in Arabidopsis thaliana |
| GSE71657 |
ChIP-chip and transcriptomic analysis of Arabidopsis BRM chromatin remodeler |
|
