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| Status |
Public on Dec 01, 2020 |
| Title |
2236F16-2: nrde3_H3K9me3_1 |
| Sample type |
SRA |
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| Source name |
early embryo
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: nrde-3(tm1116) X
tissue: early embryo
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| Growth protocol |
chromatin was isolated from early embryos (before bean stage) grown after L1 synchronization (60–65 h depending on each strain) in two independent biological replicates.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP experiments were performed as previously described (Zeller et al., 2016). In brief: Embryos were harvested from synchronized animals in three replicates. 40 μg of chromatin was incubated overnight with 3 μg of anti-LEM-2 antibody (Novus Biologicals) coupled to Dynabeads Sheep Anti-Rabbit IgG (Invitrogen), in FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl)) containing 1% SDS. Antibody bound chromatin was washed 3 × 5 min FA buffer; 5 min FA buffer with 1 M NaCl; 10 min FA buffer with 500 mM NaCl; 5 min with TEL buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0) and 2 × 5 min with TE. Complexes were eluted in 1% SDS in TE with 250 mM NaCl at 65°C for 15 min. Samples and inputs were treated with 20 μg of RNAse A for 30 min at 37°C and 20 μg of proteinase K for 1 hr at 55°C. Crosslinks were reversed by overnight incubation at 65°C. DNA was subsequently purified using Zymo DNA purification columns (Zymo Research).
Libraries were prepared from chromatin IP and input samples using the NEBNext ultra DNA library prep kit for Illumina (NEB # 7370) and the NEBNext Multiplex Oligos for Illumina (NEB # E7335), according to the manufacturers recommendations. No size selection was performed during sample preparation and the libraries were indexed and amplified using 12 PCR cycles, using the recommended conditions. After a final cleanup with Agencourt AmPure XP beads (Beckman # A63881), the library size distribution and concentrations were determined using a BioAnalyzer 2100 (Agilent technologies) and Qubit (Invitrogen) instrument, respectively. The final pools were prepared by mixing equimolar amounts of all individual indexed libraries and then sequenced on a HiSeq 2500 (Illumina) in Rapid mode (Paired-End 50).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Basecalling was performed using RTA v. 1.13.48.
Reads were aligned to the ce10 (WS220) genome assembly using the qAlign function from the QuasR R package (v. 3.2).
The qCount function in the QuasR package was used to count reads overlapping the ce6 genome assembly split into fragments of 500bp.
Genome_build: ce10 (WS220)
Supplementary_files_format_and_content: Tab-delimited file. First column contains seqnames, followed by start and end position and columns contain raw counts of reads overlapping each fragment for each sample.
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| Submission date |
Aug 20, 2020 |
| Last update date |
Dec 01, 2020 |
| Contact name |
Jan Padeken |
| E-mail(s) |
[email protected]
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| Organization name |
Institute of Molecular Biology
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| Street address |
Ackermannweg 4
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| City |
Mainz |
| ZIP/Postal code |
55128 |
| Country |
Germany |
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| Platform ID |
GPL18245 |
| Series (2) |
| GSE156548 |
Two parallel pathways recruit the H3K9me3 HMT in somatic cells, requiring the Argonaut NRDE-3, or the MBT-domain protein, LIN-61 (ChIP-seq) |
| GSE156551 |
Two parallel pathways recruit the H3K9me3 HMT in somatic cells, requiring the Argonaut NRDE-3, or the MBT-domain protein, LIN-61 |
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| Relations |
| BioSample |
SAMN15866105 |
| SRA |
SRX8976688 |
| 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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