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| Status |
Public on Dec 17, 2015 |
| Title |
H3K4me3_ip_Xins_2 |
| Sample type |
SRA |
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| Source name |
Xins cells
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| Organism |
Schmidtea mediterranea |
| Characteristics |
cell type: Xins
replicate: 2
RNAi treatment: None (WT)
chip antibody: anti-H3K4me3 (Millipore, catalog# 07-473, lot# 2459618)
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| Treatment protocol |
dsRNA-containing food for RNAi treatment was generated as previously described (Gurley et al., 2008); briefly, cloned genes were transformed into bacterial strain Ht115, induced to express dsRNA with 1mM (final) IPTG, incubated with shaking at 37C for 2 hours, then pelleted, rinsed, and mixed with calf liver paste for ingestion. Live worms images were captured using a Leica M205-FA; movies were captured using a Zeiss Lumar V12 equipped with an Axiocam HRc. Worms were subjeted to RNAi for 6 days for set RNAI and 9 days from mll RNAi
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin-immunoprecipitation was performed as previously described (Lee et al., 2006) with some modifications. Briefly, worms were snap-frozen in liquid nitrogen, homogenized with a clean razor, resuspended in freshly cracked 4% PFA (EMS)/calcium- and magnesium-free buffer with 1% BSA (CMFB) and rocked for 10 minutes at room temperature to cross-link and dissociate. Samples were then quenched with 0.125M glycine, filtered with 100μm cell strainers (Corning), pelleted by centrifugation at 700*g, and washed twice with ice-cold 0.4XPBS + 10% FBS + 1X Roche Complete protease inhibitors. FACS-isolated planarian cells and Drosophila S2 cells were cross-linked in suspension in collection buffer + 10% FBS or Schneider’s media + 10% FBS, respectively. Pellets were then either snap-frozen or resuspended in lysis buffer (1% SDS, 50mM Tris pH 8, 10mM EDTA) and incubated on ice for 10 min. Samples were then sheared for 12 min in the S220 Focused Ultrasonicator (Covaris) in milliTubes (Covaris) with either the AFA fiber or 100μl glass beads (for volumes < 1ml). Samples were then cleared by centrifugation (10K*g, 10 min) and used for immunoprecipitations. H3K4me3 antibody (Millipore 07-473) was used at 0.5-1μl antibody/μg chromatin (as estimated by Qubit measurement of dsDNA in IP inputs) and incubated 4hrs - overnight at 4°C. Antibody-chromatin complexes were then captured with a mix of ProteinA/G magnetic beads (Life Technologies; 2hrs, 4°C), washed, eluted and incubated overnight at 65°C to reverse cross-links. Samples were then digested with RNase and ProteinaseK, and DNA was isolated by column purification (Qiagen).
DNAseq libraries were made using an optimized protocol with reagents from the Illumina TruSeq RNA kit and data was collected for ChIP Seq analysis on an Illumina HiSeq 2500.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
diffReps_output_X1_Xins.txt
Xins_2_peaks.bed
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| Data processing |
Base calls were performed using CASAVA-1.8.2
Single-end 51 base reads were mapped to version 3.1 of the S. mediterranea genome using bowtie with the following parameters: best --strata v 2 m 2 .
H3K4me3 difference peaks between control (unc22(RNAi)) and knockdown (set1(RNAi) or mll1/2(RNAi)) were called using diffReps (Shen et al., 2013) with the following parameters: --btr --bco --nsd sharp --frag 250. Peaks were mapped to genes and all further quantitative analysis was carried out using R and Bioconductor.
Peaks were found using macs2 with the following parameters: -g 895484558 -q 0.01 --model. The "v31.xxxxxx" identifiers represent contigs.
Genome_build: Schmidtea_mediterranea_3.1
Supplementary_files_format_and_content: diffReps output files.
Supplementary_files_format_and_content: MACS2 peaks files.
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| Submission date |
Oct 19, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Alex Daniel Chitsazan |
| E-mail(s) |
chitsazanalex@gmail.com
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| Phone |
5039169064
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| Organization name |
Oregon Health and Science University
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| Street address |
2720 S Moody Ave
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| City |
Happy Valley |
| State/province |
OR |
| ZIP/Postal code |
97239 |
| Country |
USA |
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| Platform ID |
GPL20150 |
| Series (2) |
| GSE74153 |
Set1 and MLL1/2 target distinct sets of functionally different genomic loci in vivo: An in depth ChIP-seq analysis of H3K4me3 targets in vivo and upon Set1 and mll1 knockdown |
| GSE74169 |
Set1 and MLL1/2 target distinct sets of functionally different genomic loci in vivo |
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| Relations |
| BioSample |
SAMN04194351 |
| SRA |
SRX1354640 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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