 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 23, 2012 |
| Title |
larvae_mb_wce |
| Sample type |
SRA |
| |
|
| Source name |
whole larvae
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: 3rd instar larvae
antibody: none
ChIP: NONE
genotype/variation: Jarid2 mutant
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
1g of larvae were homogenized (TISSUE TEAROR, model 985370-395, BIOSPEC PRODUCTS, INC.) on low speed for 2 min in 10 ml of buffer A1 (15 mM HEPES pH7.5, 15 mM NaCl, 60 mM KCl, 4 mM MgCl2, 0.5% Triton X-100, 0.5 mM DTT, protease inhibitors (Roche, complete, EDTA-free, Cat No. 1873 580)) plus 1.3 ml of 16% paraformaldehyde (1.8% final) following further homogenization with a dounce tissue grinder 15 times with a loose pestle (Wheaton, 15 ml dounce). The sample was incubated on a nutator at room temperature until 15 min after the first homogenization step and then quenched with 1130 μl of 2.5 M glycine (225 mM glycine final) for 5 min at room temperature. The homogenate was passed through a 70 μm cell strainer (FALCON, 2350) and centrifuged for 5 min at 2000g, 4ºC. The supernatant was aspirated, resuspended in 5 ml of buffer A1 and centrifuged for 5 min at 2000g, 4ºC. The supernatant was aspirated again, resuspended in 5 ml of buffer A2 (10 mM Tris HCl pH 7.5, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100, 0.5 mM DTT, protease inhibitors (Roche, complete, EDTA-free, Cat No. 1873 580)) and centrifuged for 5 min at 2000g, 4ºC. The previous step was repeated one more time and the pellet resuspended in 4 ml RIPA buffer (25 mM Tris pH 7.5, 140 mM NaCl, 1% Triton X-100, 1 mM EDTA, 0.1% SDS, 0.1% Na-deoxycholate, 0.5 mM DTT, protease inhibitors (Roche, complete, EDTA-free, Cat No. 1873 580)) with 0.5% N-lauroylsarcosine. 1.3 ml samples were biorupted (Diagenode, Bioruptor) two times for 15 min (50% on/off) in 15 ml hard plastic tubes (Corning, 430055) following centrifugation for 20 min at 14 krpm, 4ºC. The supernatant was kept and 10 μl of biorupted chromatin reserved for gel analysis (to check sizing) and 150 μl as input control. The sizing sample was reverse cross-linked at 65ºC over night with 90 μl TE and 3 μl proteinase K (30 mg/μl) and the input sample with 5 μl proteinase K. The remaining chromatin was diluted five fold with RIPA buffer and incubated over night with the respective antibody on a nutator at 4ºC. 60 μl of protein A agarose (Invitrogen, 15918-014) was washed in 5 ml RIPA buffer, centrifuged for 2 min at 1000 rpm, 4ºC. The supernatant was aspirated, the chromatin sample added and incubated for 2 h on a nutator at 4ºC. After centrifugation for 2 min at 1000 rpm, 4ºC the protein A agarose was transferred into a 1.5 ml tube, washed with 1 ml RIPA buffer, incubated for 5 min on a nutator at room temperature and centrifuged for 2 min at 2500 rpm, 4ºC. The supernatant was aspirated and the previous washing steps repeated another seven times. Elution was performed on a nutator at room temperature for 20 min with 300 μl elution buffer (0.1 M NaHCO3, 1% SDS) containing proteinase K (1 ml elution buffer and 5 μl proteinase K (30 mg/μl) and the sample centrifuged for 2 min at 2500 rpm at room temperature. The supernatant was kept and the elution step repeated. Elution fractions were pooled and reverse cross-linked at 65ºC over night. DNA was isolated with the Qiagen PCR purification kit and eluted in 50 μl H2O and DNA concentration determined by Pico Green Assay. 20 ng of DNA was amplified (Single end primers, Illumina) and analyzed on a 2100 Bioanalyzer (Agilent Technologies) before submitted to sequencing (Genome Analyzer, Illumina). ChIP-sequencing of eye-antenna imaginal discs was performed following (Papp and Muller 2006). Samples were biorupted and processed as described above.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Filtered reads were then aligned to the fly genome (UCSC dm3) using the bowtie (Langmead et al., 2009) alignment tool, version 0.12.7. Only those sequences that matched uniquely to the genome with up to two mismatches were retained for subsequent analysis. Enriched regions of ChIP-seq signal for Jarid2 and Su(z)12 were determined by the ‘MACS’ (Zhang et al., 2008) peak-finding program, version 1.4.0rc2. Sequence reads for each ChIP-seq dataset and their associated whole-cell extract controls were used for the input and control file, respectively. The effective genome size was configured appropriately for the fly datasets, and the p-value cutoff was set to 1.00e-08 or FDR < 1%, and a fold-change greater than four. All other MACS parameters were left default.
|
| |
|
| Submission date |
Nov 08, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Alexander (Garrett) Garruss |
| E-mail(s) |
[email protected]
|
| Organization name |
Stowers Institute for Medical Research
|
| Lab |
Shilatifard
|
| Street address |
1000 East 50th Street
|
| City |
Kansas CIty |
| State/province |
Missouri |
| ZIP/Postal code |
64110 |
| Country |
USA |
| |
|
| Platform ID |
GPL11203 |
| Series (2) |
| GSE33546 |
Polycomb repressive complex 2-dependent and -independent functions of Jarid2 in transcriptional regulation in Drosophila [ChIP-Seq] |
| GSE36039 |
Polycomb repressive complex 2-dependent and –independent functions of Jarid2 in transcriptional regulation in Drosophila |
|
| Relations |
| SRA |
SRX104975 |
| BioSample |
SAMN00750675 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM829679_mb_wce.txt.bam.bed.gz |
215.8 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
