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| Status |
Public on Oct 12, 2022 |
| Title |
C37 dRING Rep 2 |
| Sample type |
SRA |
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| Source name |
Schneider 2 (S2) cells
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain: Oregon R
developmental stage: Late embryonic stage
antibody: rabbit anti-dRING (from Yuri Schwartz)
spike-in: Drosophila virilis
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| Treatment protocol |
Experiments were conducted on cells at 80-90% confluency. Cells were resuspended in FBS-free media and incubated with 30 μM of C646 or the inactive analog C37 for 30 minutes.
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| Growth protocol |
S2 cells were cultured at 25°C in Schneider's Drosophila medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% Penicillin-Streptomycin (Gibco).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed in 1% formaldehyde (Sigma Aldrich, F8775) for 15 min at room temperature, quenched with 0.16 M glycine for 5 min at 4°C and washed in cold PBS. Crude nuclear extracts were obtained by sequentially washes with ChIP buffer A (10 mM HEPES pH 7.6, 10 mM EDTA pH 8.0, 0.5mM EGTA pH 8.0 and 0.25% Triton X-100) followed by ChIP buffer B (10mM HEPES pH 7.6, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0 and 0.01% Triton X-100) to isolate nuclei. Nuclei were resuspended in Sonication buffer (15 mM HEPES pH 7.6, 140 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 0.1% sodium deoxycholate and 1% Triton X-100), with Complete Mini EDTA-Free protease inhibitor tablets, Roche) supplemented with 0.5% SDS and 0.2% n-lauroylsarcosine, at a final concentration of 5 – 10 x 107 cells/ml and sonicated in a Bioruptor (Diagenode) using high power settings to obtain an average fragment size distribution of 200–500 bp. The concentration of chromatin was quantified and spike-in chromatin from Drosophila virilis cells was added. Immunoprecipitations were performed by incubating 10 μg of chromatin with anti-CBP (homemade), anti-dRING (kind gift of Yuri Schwartz, Umeå Universitet, Sweden), anti-E(z) (kind gift of Richard Jones, Southern Methodist University, USA) and anti-Pho (kind gift of Judith Kassis, National Institute of Health, USA) overnight. Chromatin-antibody complexes were bound to Protein A and G Dynabeads (Invitrogen) and washed sequentially with Wash A (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 140 mM NaCl, 0.1% SDS, 0.1% Sodium Deoxycholate and 1% Triton X-100), Wash B (Wash A adjusted to 500 mM NaCl), Wash C (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5% Sodium Deoxycholate and 0.5% IGEPAL CA-630) and Tris-EDTA (TE). Beads were resuspended in 100 μl TE, treated with RNase A (20 μg/ml) at 55°C for 30 min, supplemented with SDS (0.75%) and Tris (50 mM). and crosslinks reversed at 65°C overnight. Eluted ChIP DNA was treated with Proteinase K at 55°C for 2 h and purified using the ChIP DNA Clean & Concentrator kit (ZymoResearch, D5205).
Libraries were constructed from ChIP samples (2-5 ng) with the NEBNext Ultra II DNA Library Prep Kit (NEB) and single-end (1x75 bp) sequenced on an Illumina NextSeq 550 platform at the BEA core facility, Stockholm.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 550 |
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| Description |
C37 dRING Rep 2
C37_R2_dRING_S11
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| Data processing |
ChIP-seq reads from each sequenced library were mapped to the Drosophila melanogaster (dm6) genome assembly using Bowtie2 with the default parameters. Duplicated and unmapped reads were removed using Picard. To obtain the spike-in reads, we mapped the reads to the Drosophila virilis (dvir_caf1) genome assembly and the D. melanogaster reads were normalized based on the ratio of D. virilis reads. From the spike-in adjusted data, RPKM normalized ChIP-seq coverage tracks were generated using the deepTools. Bigwig files of the mean RPKM were produced using the deepTools comparebigwig tool.
Genome_build: dm6
Supplementary_files_format_and_content: bigwig for coverage
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| Submission date |
Apr 28, 2021 |
| Last update date |
Oct 12, 2022 |
| Contact name |
Mattias Mannervik |
| E-mail(s) |
mattias.mannervik@su.se
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| Organization name |
Stockholm University
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| Department |
Molecular Biosciences, the Wenner-Gren Institute
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| Lab |
Mattias Mannervik
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| Street address |
Arrheniuslaboratories E3
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| City |
Stockholm |
| ZIP/Postal code |
10691 |
| Country |
Sweden |
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| Platform ID |
GPL22106 |
| Series (2) |
| GSE173473 |
p300/CBP sustains Polycomb silencing by non-enzymatic functions (ChIP-seq on inhibitor-treated Drosophila S2 cells) |
| GSE186723 |
p300/CBP sustains Polycomb silencing by non-enzymatic functions |
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| Relations |
| BioSample |
SAMN18902398 |
| SRA |
SRX10695070 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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