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| Status |
Public on Dec 04, 2010 |
| Title |
[E-MTAB-214] NSL1 |
| Sample type |
SRA |
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| Source name |
NSL1
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| Organism |
Drosophila melanogaster |
| Characteristics |
material type: organism_part
developmentalstage: 3rd instar larvae
organismpart: salivary_glands
chip antibody: NSL1
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| Growth protocol |
grow | Chromatin was obtained from male 3rd instar larvae salivary glands as previously described (Legube et al, 2006). In brief, collected salivary glands (10 per ChIP) were fixed in 1 mL of fixing solution (50 mM HEPES at pH 7.6, 100 mM NaCl, 0.1 mM EDTA at pH 8, 0.5 mM EGTA at pH 8, 2% formaldehyde) for 15 min at room temperature on a rotating wheel. The glands were centrifuged at 2000 rpm for 1 min, washed once in PBS with 0.01% Triton X-100 and 0.125 M glycine; then washed for 10 mins each in 1 ml of buffer A (0.25% Triton X-100, 10 mM EDTA (pH 8), 0.5 mM EGTA (pH 8), 10 mM Tris (pH 8)) and buffer B (200 mM NaCl, 10 mM Tris (pH 8), 10 mM EDTA (pH 8), and 0.5 mM EGTA (pH 8)). The glands where then resuspended in 500 _L of sonication buffer (10 mM Tris at pH 8, 1 mM EDTA at pH 8, 0.5 mM EGTA at pH 8.0) and sonicated 8 min (pulsed eight times for 30 sec, paused for 30 sec, high) in a Bioruptor (Cosmo Bio).
specified_biomaterial_action | Immunoprecipitation was then performed on 500 _L of chromatin from salivary glands, according to Orlando et al. (1997), using 3 _L of a polyclonal antibody against NSL-1, MCRS2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
nucleic_acid_extraction | Chromatin was obtained from male 3rd instar larvae salivary glands as previously described (Legube et al, 2006). In brief, collected salivary glands (10 per ChIP) were fixed in 1 mL of fixing solution (50 mM HEPES at pH 7.6, 100 mM NaCl, 0.1 mM EDTA at pH 8, 0.5 mM EGTA at pH 8, 2% formaldehyde) for 15 min at room temperature on a rotating wheel. The glands were centrifuged at 2000 rpm for 1 min, washed once in PBS with 0.01% Triton X-100 and 0.125 M glycine; then washed for 10 mins each in 1 ml of buffer A (0.25% Triton X-100, 10 mM EDTA (pH 8), 0.5 mM EGTA (pH 8), 10 mM Tris (pH 8)) and buffer B (200 mM NaCl, 10 mM Tris (pH 8), 10 mM EDTA (pH 8), and 0.5 mM EGTA (pH 8)). The glands where then resuspended in 500 _L of sonication buffer (10 mM Tris at pH 8, 1 mM EDTA at pH 8, 0.5 mM EGTA at pH 8.0) and sonicated 8 min (pulsed eight times for 30 sec, paused for 30 sec, high) in a Bioruptor (Cosmo Bio). The fragments generated were 300 bp on an average. Immunoprecipitation was then performed on 500 _L of chromatin from salivary glands, according to Orlando et al. (1997), using 3 _L of a polyclonal antibody against NSL-1, MBD-R2, MCRS2 and MOF or a correspondent preimmune anti-serum (mock ChIP). The immunoprecipitated DNA was resuspended in 100 ml of nuclease free water. Since the ChIP derived DNA was already sufficiently fragmented, we did not perform a DNA fragmentation step.
sequencing | Sequencing was carried out using the 1G Illumina Genome Analyser (Solexa) according to the manufacturer's instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
Performer: EMBL_Heidelberg
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| Data processing |
processed data not provided
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| Submission date |
Nov 30, 2010 |
| Last update date |
May 15, 2019 |
| Organization |
European Bioinformatics Institute |
| E-mail(s) |
miamexpress@ebi.ac.uk
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| Lab |
ArrayExpress
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| Street address |
Wellcome Trust Genome Campus
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| City |
Hinxton |
| State/province |
Cambridgeshire |
| ZIP/Postal code |
CB10 1SD |
| Country |
United Kingdom |
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| Platform ID |
GPL9058 |
| Series (1) |
| GSE25709 |
[E-MTAB-214] ChiP-seq profiling of Drosophila melanogaster salivary glands to identify targets for NSL1 and MCRS2. |
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| Relations |
| SRA |
ERX004312 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data not provided for this record |
| Raw data are available in SRA |
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