NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM829681 Query DataSets for GSM829681
Status Public on Feb 23, 2012
Title larvae_mb_h3k27me3
Sample type SRA
 
Source name whole larvae
Organism Drosophila melanogaster
Characteristics tissue: 3rd instar larvae
antibody: rb-α-H3K27me3 (Active Motif, 39155)
ChIP: H3K27me3
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 SRX104977
BioSample SAMN00750677

Supplementary file Size Download File type/resource
GSM829681_mb_h3k27me3.txt.bam.bed.gz 250.1 Mb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap