|
| Status |
Public on Jun 28, 2017 |
| Title |
Ser2P Mock Replicate 1 (chip-seq) |
| Sample type |
SRA |
| |
|
| Source name |
ML-DmBG3-c2 cultured cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
RNAi treatment: Mock RNAi treatment
chip antibody source: Phospho-Rpb1 CTD Ser2P (#13499, Cell Signaling)
input file for normalization: Input_Replicate 1 and Input_Replicate 2 fastqs
|
| 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 3 hours. For RNAi of all factors, from 0.7 to 40 μg of dsRNA was added per well of a 6-well plate. Media was adjusted to 3 ml and 10% FCS with Schneider'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.
|
| 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 5E6 per 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 1x10^9/3 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 ~50x10^6 cells, were snap frozen on dry ice. For immunoprecipitation, aliquots of chromatin (10 - 20 x 10^6 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 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 or phenol/choloroform extraction and ethanol precipitation.
Ion Torrent IonXpress barcoded sequencing libraries were constructed using the Ion Xpress Plus Fragment Library Kit (Life Technologies) according to the manufacturer's directions except that size selection was performed using Beckman Agencourt magnetic beads.
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| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Ion Torrent Proton |
| |
|
| Description |
Ser2P.BG3.mock.ave.log.enrichment.sgr
|
| Data processing |
Basecalling was performed by Torrent Suite version 4 and 5 software using default settings
Aligmentment to the Drosophila release 5 genome sequence was performed using the TMAP map4 algorithm without soft clipping (-g 3 option)
Genome coverage at each base pair for Input and each ChIP sample was calcluted using BEDtools genomecoveragebed (-d option)
Enrichment in ChIP sample coverage relative to Input coverage in sliding 250 bp windows with 50 bp steps was calculated using custom R scripts as described in Swain et al. submitted. Input file used for each sample is indicated above. Coverage in each window was normalized to total genome coverage prior to normalization to Input.
ChIP enrichment was averaged for all replicates to generate final processed data files
Genome_build: Drosophila melanogaster Release 5 (April 2006) with ChrU and Uextra removed
Supplementary_files_format_and_content: Text graph file (sgr) of log2 enrichment normalized to Input
|
| |
|
| Submission date |
Jun 27, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Dale Dorsett |
| Organization name |
Saint Louis University School of Medicine
|
| Department |
Biochemistry and Molecular Biology
|
| Street address |
1100 South Grand Boulevard
|
| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63104 |
| Country |
USA |
| |
|
| Platform ID |
GPL19528 |
| Series (2) |
| GSE100544 |
Polycomb Repressive Complex 1 regulates transcription of active genes [ChIPseq] |
| GSE100548 |
Polycomb Repressive Complex 1 regulates transcription of active genes |
|
| Relations |
| BioSample |
SAMN07281252 |
| SRA |
SRX2960456 |
