|
| Status |
Public on Apr 24, 2013 |
| Title |
Rpb3_BG3_Rad21RNAi |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Rpb3 ChIP from Rad21 RNAi-treated Drosophila ML-DmBG3-c2 cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
gender: male
antibody: Anti-Rpb3, rabbit (Karen Adelman, NIEHS, NIH)
cell type: ML-DmBG3-c2
treatment: Rad21 RNAi-treated
|
| Treatment protocol |
For RNAi, media was replaced with 1 ml of Express Five SFM (Invitrogen) with 1% FCS, (and 10 μg per ml insulin for BG3 cells), for 2 hours. 10 μg of dsRNA was added per well of a 6-well plate. Media was adjusted to 3 ml and 10% FCS withSchneider's media after 2 hrs of RNAi treatment. Cells were replated as needed. Templates for dsRNA synthesis were made by PCR from cDNA or genomic DNA templates using primers with T7 promoters. In most experiments, equal amounts of two dsRNAs against each target were used. Both individual dsRNAs knocked down the targets, but knockdown was generally more efficient with a mixture. Templates were designed to avoid off-target matches of ≥19 nucleotides using Drosophila RNAi Screening Center (http://www.flyrnai.org/) online tools. Cohesin RNAi-treated cells were screened for sister chromatid cohesion defects, and none were found, though a mild cell division delay occurred immediately following RNAi treatment.
|
| Growth protocol |
BG3 cells were cultured in Schneider's media with 10% FCS and 10 μg per ml insulin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
BG3 cells were grown to a concentration of 5x106/mL, then crosslinked by addition of 36% formaldehyde directly to the cell culture media to a final concentration of 1%, and incubation for 10 minutes at room temperature on a rocking platform. The reaction was stopped by addition of glycine pH 7.0 to a final concentration of 0.125 M. Cells were washed once with 1x PBS and once each with Wash Buffers A (10 mM HEPES pH 7.6; 10 mM EDTA pH 8.0; 0.5 mM EGTA pH 8.0; 0.25% Triton X100) and B (10 mM HEPES pH 7.6; 200 mM NaCl; 1 mM EDTA pH 8.0; 0.5 mM EGTA pH 8.0; 0.01% Triton X-100) for 10 minutes each. Cells were resuspended in sonication buffer to a concentration of 1x109/3 mL, and sonicated using a BioRuptor Sonicator for 14-20 cycles of 30 s on, 30 s off. Na-lauroylsarcosine was added to 0.5% and incubated for 10 minutes, rotating. Chromatin was centrifuged at 12,000g for 5 minutes at 4°C to pellet insoluble material, and 200 uL chromatin aliquots, each representing ~50x106 cells, were snap frozen on dry ice. For immunoprecipitation, 200 μL aliquots of chromatin (50 x 106 BG3 cells) were adjusted to RIPA buffer in a final volume of 500 μL. Adjusted chromatin was pre-cleared for 2 hours with Protein A agarose beads, then incubated with primary antibody overnight at 4°C. Antibody-chromatin complexes were pulled down by incubation with Protein A/G agarose beads for 4 hours at 4°C, and beads were washed consecutively with RIPA, LiCl buffer, and TE. Samples were treated with RNAse A for 30 minutes at 37°C, then DNA crosslinks were reversed by overnight treatment at 65°C with Proteinase K, and DNA was recovered using Qiagen MinElute columns.
|
| Label |
biotin
|
| Label protocol |
Immunoprecipitated DNA was amplified using the GenomePlex Complete Whole Genome Amplification Kit (Sigma) and fragmented using controlled DNAse I reactions. Fragmented DNA was labeled with bio-11-ddATP (Perkin Elmer) using a Terminal Transferase labeling kit (Roche).
|
| |
|
| Channel 2 |
| Source name |
Input DNA from ML-DmBG3-c2 cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
gender: male
antibody: none
cell type: ML-DmBG3-c2
|
| Treatment protocol |
For RNAi, media was replaced with 1 ml of Express Five SFM (Invitrogen) with 1% FCS, (and 10 μg per ml insulin for BG3 cells), for 2 hours. 10 μg of dsRNA was added per well of a 6-well plate. Media was adjusted to 3 ml and 10% FCS withSchneider's media after 2 hrs of RNAi treatment. Cells were replated as needed. Templates for dsRNA synthesis were made by PCR from cDNA or genomic DNA templates using primers with T7 promoters. In most experiments, equal amounts of two dsRNAs against each target were used. Both individual dsRNAs knocked down the targets, but knockdown was generally more efficient with a mixture. Templates were designed to avoid off-target matches of ≥19 nucleotides using Drosophila RNAi Screening Center (http://www.flyrnai.org/) online tools. Cohesin RNAi-treated cells were screened for sister chromatid cohesion defects, and none were found, though a mild cell division delay occurred immediately following RNAi treatment.
|
| Growth protocol |
BG3 cells were cultured in Schneider's media with 10% FCS and 10 μg per ml insulin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
BG3 cells were grown to a concentration of 5x106/mL, then crosslinked by addition of 36% formaldehyde directly to the cell culture media to a final concentration of 1%, and incubation for 10 minutes at room temperature on a rocking platform. The reaction was stopped by addition of glycine pH 7.0 to a final concentration of 0.125 M. Cells were washed once with 1x PBS and once each with Wash Buffers A (10 mM HEPES pH 7.6; 10 mM EDTA pH 8.0; 0.5 mM EGTA pH 8.0; 0.25% Triton X100) and B (10 mM HEPES pH 7.6; 200 mM NaCl; 1 mM EDTA pH 8.0; 0.5 mM EGTA pH 8.0; 0.01% Triton X-100) for 10 minutes each. Cells were resuspended in sonication buffer to a concentration of 1x109/3 mL, and sonicated using a BioRuptor Sonicator for 14-20 cycles of 30 s on, 30 s off. Na-lauroylsarcosine was added to 0.5% and incubated for 10 minutes, rotating. Chromatin was centrifuged at 12,000g for 5 minutes at 4°C to pellet insoluble material, and 200 uL chromatin aliquots, each representing ~50x106 cells, were snap frozen on dry ice. For immunoprecipitation, 200 μL aliquots of chromatin (50 x 106 BG3 cells) were adjusted to RIPA buffer in a final volume of 500 μL. Adjusted chromatin was pre-cleared for 2 hours with Protein A agarose beads, then incubated with primary antibody overnight at 4°C. Antibody-chromatin complexes were pulled down by incubation with Protein A/G agarose beads for 4 hours at 4°C, and beads were washed consecutively with RIPA, LiCl buffer, and TE. Samples were treated with RNAse A for 30 minutes at 37°C, then DNA crosslinks were reversed by overnight treatment at 65°C with Proteinase K, and DNA was recovered using Qiagen MinElute columns.
|
| Label |
biotin
|
| Label protocol |
Immunoprecipitated DNA was amplified using the GenomePlex Complete Whole Genome Amplification Kit (Sigma) and fragmented using controlled DNAse I reactions. Fragmented DNA was labeled with bio-11-ddATP (Perkin Elmer) using a Terminal Transferase labeling kit (Roche).
|
| |
|
| |
| Hybridization protocol |
Hybridization cocktails were generated using the labeled DNA fragments (2 μg labeled DNA, 1x MES, 3M TMAC, 40 pM Affymetrix control oligo B2, 100 μg/mL salmon sperm DNA, and 0.02% Triton X-100). Each chip was pre-hybridized with 200 μl of 1X MES-triton for 1 hour in the Affymetrix Hybridization 645 oven at 45 rpm and 45oC. Samples in hybridization cocktail were heated for 10 minutes at 99oC then incubated to a 45oC for 10 minutes. Samples are spun in a microcentrifuge at maximum speed for 3 min. 200 μl of a sample was injected in to a chip. Hybridization was carry out in the hybridization oven at 45 rpm and 45oC for 18 hours. The arrays were washed and stained on the GeneChip Fluidics Station 450 series.
|
| Scan protocol |
Arrays were scanned on an Affymetrix GeneChip® Scanner 3000 7G.
|
| Description |
Rpb3 ChIP from Rad21 RNAi-treated Drosophila ML-DmBG3-c2 cells
|
| Data processing |
MAT software (Johnson et al. 2006) was used to calculate ChIP enrichment and significance across the non-repetitive Drosophila genome.
matscore.bar files contain MATscores for each probe as generated using MAT software (Johnson et al. 2006). .bed files indicate significant binding at p-value less than or equal to 10-3.
|
| |
|
| Submission date |
Nov 16, 2012 |
| Last update date |
Apr 24, 2013 |
| Contact name |
Cheri A Schaaf |
| E-mail(s) |
[email protected]
|
| Phone |
314-977-9224
|
| Organization name |
Saint Louis University School of Medicine
|
| Department |
Biochemistry & Molecular Biology
|
| Lab |
Dorsett
|
| Street address |
1100 South Grand Ave
|
| City |
St Louis |
| State/province |
MO |
| ZIP/Postal code |
63104 |
| Country |
USA |
| |
|
| Platform ID |
GPL6629 |
| Series (2) |
| GSE42360 |
Genome-wide control of RNA polymerase II activity by cohesin (tiling) |
| GSE42399 |
Genome-wide control of RNA polymerase II activity by cohesin |
|
