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| Status |
Public on Apr 13, 2011 |
| Title |
MOF_RIP_FA |
| Sample type |
RNA |
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| Source name |
MOF_RIP_FA
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: S2 cells (ATCC CRL-1963)
antibody: MOF
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| Treatment protocol |
The cells were pelleted, washed 2 times in 100 ml 10 mM HEPES pH 7.4, 140 mM NaCl and resuspended in 50 ml of lysis buffer (20 mM HEPES pH 7.4, 3 mM MgCl2, 0.1% Triton X-100, 1 mM dithioreitol, 0.5mM PMSF, 10 U/ml RNasin and 0.5 × Protease inhibitor cocktail (Roche)).The cells were allowed to swell on ice for 10 minutes and then homogenized on ice with 30 strokes of a Dounce homogenizer. The nuclei were pelleted at 2,000 × g for 5 minutes and used either as native (non-crosslinked) or after formaldehyde crosslinking. For the crosslinked sample (FA) the pelleted nuclei were resuspended in 50 ml lysis buffer and cross-linked by adding formaldehyde to a final concentration of 0.5% and incubated for 10 minutes in room temperature. The crosslinking was stopped by adding glycine (final concentration 0.125M), the nuclei were washed once in lysis buffer and resuspended in 2 ml of sonication buffer (20 mM HEPES pH 7.4, 10% glycerol, 0.1M NaCl, 1 mM MgCl2, 0.1% Triton X-100, 1 mM dithioreitol, 0.5mM PMSF, 10 U/ml RNasin and 0.5 × Protease inhibitor cocktail) and sonicated using a Bioruptor (Diagenode) for 4 minutes setting high (30 sec ON, 30 sec OFF). For the native samples (N1, N3, N6) the pelleted nuclei were resuspended in 2 ml sonication buffer and sonicated for 1, 3 or 6 minutes (N1, N3 and N6, respectively). Cellular debris were removed by centrifugation for 25 minutes, 4°C at 14,000 × g and the supernatants were used for immunoprecipitations.
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| Growth protocol |
For each sample treatment condition we used 300ml D. melanogaster Schneider’s line 2 cells (ATCC CRL-1963) grown at 25°C in an Erlenmeyer flasks at a density of 1×10^7 cells/ml in Drosophila SFM medium (Invitrogen) supplemented with 100U/ml Penicillin G, 100µg/ml Streptomycin sulfate and 2mM of L-glutamine.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For immunoprecipitation, 5-10 mg nuclear extracts were incubated with 5 µl anti-MSL2, 5 µl anti-MOF for 45 minutes at 4°C with agitation. The antibody complexes were precipitated by incubation with 75 µl of Dynabeads conjugated to protein-A (Dynal) for 30 minutes at 4°C with agitation. The supernatant was removed and the beads were washed twice with PBS (150 mM NaCl), 0.1% Triton X-100, 32 U/ml RNasin, 0.5 × Protease inhibitor cocktail and twice in PBS (300 mM NaCl), 0.1% Triton X-100, 32 U/ml RNasin, 0.5 × Protease inhibitor cocktail. The crosslinking in sample FA were reversed by adding 200 µl 0.45 M LiCl to the beads and incubate 3-4 hours at 65°C. RNA were isolated using TRI-reagent (Ambion) followed by a purification using RNeasy kit (Qiagen) according to the instruction by the suppliers. The RNA samples were concentrated and reverse transcribed into cDNA using random primers with the ImPromII first strand synthesis kit (Promega) according to the suppliers recommendations. The single stranded cDNA was purified with QIAquick PCR purification Kit (Qiagen). The purified cDNA samples were amplified using WGA2 GenomePlex Complete whole genome amplification kit (Sigma) according to the recommendations by the supplier.
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| Label |
Biotin
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| Label protocol |
Samples were prepared and hybridized to GeneChip Drosophila Tiling 1.0R Array according to Affymetrix specifications.
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| Hybridization protocol |
Samples were prepared and hybridized to GeneChip Drosophila Tiling 1.0R Array according to Affymetrix specifications.
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| Scan protocol |
Affymetrix procedures followed
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| Description |
MOF_FA.CEL
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| Data processing |
The signal intensity data was analyzed with the Affymetrix Tiling Analysis Software (v. 1.1.02) using parameters: one side upper, 90 bp bandwidth and perfect match only. For absolute amount (transcript profile) the bandwidth was set to 0.
The resulting CHP files are generated by the Affymetrix Tiling Analysis Software (v. 1.1.02) using parameters: one side upper, 90 bp bandwidth and perfect match only. For absolute amount (transcript profile, i.e., Input_TP_bw0_N6_signal.chp) the bandwidth was set to 0.
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| Submission date |
Feb 09, 2010 |
| Last update date |
Apr 14, 2011 |
| Contact name |
Jan Larsson |
| E-mail(s) |
[email protected]
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| Organization name |
Umea University
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| Department |
Molecular Biology
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| Lab |
Jan Larsson
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| Street address |
Umea University
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| City |
Umea |
| ZIP/Postal code |
SE-90187 |
| Country |
Sweden |
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| Platform ID |
GPL6629 |
| Series (2) |
| GSE20249 |
Genome-wide profiling of RNA associated to the MSL-complex in Drosophila melanogaster |
| GSE28519 |
Pof mutant |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM507474_MOF_FA.CEL.gz |
24.6 Mb |
(ftp)(http) |
CEL |
| GSM507474_MOF_vs_Input_bw90_FA_signal.chp.gz |
15.6 Mb |
(ftp)(http) |
CHP |
| GSM507474_MOF_vs_Input_bw90_FA_signal.chp.txt.gz |
17.2 Mb |
(ftp)(http) |
TXT |
| Processed data provided as supplementary file |
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