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| Status |
Public on Jun 07, 2024 |
| Title |
dot-1.1(knu339);ced-3 mut H3K9me2 ChIP-seq repl 2 |
| Sample type |
SRA |
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| Source name |
Third larval stage animals
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| Organism |
Caenorhabditis elegans |
| Characteristics |
genotype: dot-1.1(knu339); ced-3(n1286)
antibody: H3K9me2
antibody catalog#: ab1220
antibody manufacturer: Abcam
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| Growth protocol |
Strains were maintained at 20°C under standard conditions. Hypochlorite-synchronized L1 stage animals were grown for 36 hours post-hatching at 20ºC on standard NGM plates with OP-50 E. coli.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Synchronized L3 larvae were washed off the plates using isotonic M9 solution. Fixation was performed for 30 min at 20°C in crosslinking solution (2% paraformaldehyde in M9 buffer), and excess formaldehyde was quenched with 0.1 M Tris-HCl pH 7.5. The pellet was then washed twice with M9 buffer at 4°C. The worm pellet was resuspended in 1 mL ice-cold RIPA buffer (10 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM EDTA, 0.15 M NaCl) supplemented with Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific). The suspension was then transferred to a Covaris milliTUBE 1 mL with AFA® fiber and shearing was performed using the following settings in SonoLab7: Peak Incident Power (PIP) = 240W; Duty Factor = 20%; Cycles per burst (cpb) = 200; time = 480 seconds. After shearing, the crude extract was spun for 10 min at 10,000 x g and the supernatant was collected. Protein concentration was determined by the Bradford method. Extract corresponding to 1 mg protein was diluted to 500 μl with ice-cold RIPA buffer supplemented with Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific). Antibody specific for H3K9me2 (Abcam, ab1220, 5 μg) was added and, after incubation at 4°C for 1 h, 50 μl of Dynabeads™ Protein G suspension (Thermo Fisher Scientific) were added and the mixture was incubated at 4°C for 1 h on rotation. Beads were recovered with a Dynal® Magnetic Particle Concentrator (Invitrogen) and washed at 4°C with: 0.5 mL buffer TSE-150 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), 2 X 0.5 mL buffer TSE-500 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), 0.5 mL buffer TSE-1M (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 1 M NaCl), 0.5 mL buffer LiCl (250 mM LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1), 2 X 0.5 mL buffer TE (10 mM Tris pH 8.0, 1 mM EDTA). Antibody-bound chromatin was recovered in 2 X 0.2 mL elution buffer (1% SDS, 0.1 M NaHCO3) by shaking at room temperature for 15 min. Elution buffer was added to input samples (5%) to a total volume of 0.4 mL. Input and ChIP chromatin samples were subsequently processed in parallel. After 20 μl of 5 M NaCl was added to each sample, cross-links were reversed overnight at 65°C. 10 μl of 0.5 M EDTA, 20 μl of 1 M Tris-HCl pH 6.5 and 0.5 μl of 20 mg/mL Proteinase K (Thermo Fisher Scientific) were added and each sample was incubated for 1 h at 42°C. DNA was recovered using the QIAquick PCR Purification Kit (Qiagen) in accordance with the manufacturer's instructions.
Immunoprecipitated DNA and input libraries were prepared using TruSeq ChIP Library Preparation kit following the manufacturer's instructions (Illumina) and checked for concentration and quality on DNA chips with a Bioanalyzer (Agilent). 75-bp single read sequences were generated on the NextSeq 500 sequencer according to manufacturer’s instructions (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
ChIP sample for H3K9me2 ChIP-seq in dot-1.1;ced-3 mutant, replicate 2.
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| Data processing |
Libraries were sequenced on a NextSeq500 Illumina sequencer and FASTQ files were downloaded from Illumina BaseSpace.
Reads were mapped to the C. elegans genome (ce10 assembly) using QuasR (version 1.16.0).
Read counting in regions was performed with package GenomicAlignments (version 1.16.0), using only reads with mapping quality 20 or higher. Regions without reads in all samples were removed.
Counts were normalized using the TMM method, which takes RNA composition bias into account, using the edgeR package (version 1.16.0).
For each region, coverage was first expressed as RPKM (reads per kilobase per million mapped) and log2-transformed, and then the value in the non-immunoprecipitated (input) DNA was subtracted from that in the immunoprecipitated DNA sample. Only regions in which the normalized count value in the immunoprecipitated DNA sample was higher than that in the corresponding input DNA in at least one sample in the set were considered.
Genome_build: ce10
Supplementary_files_format_and_content: Coverage files in bedGraph format were generated by counting reads at 25 bp bins. Counts were normalized using the TMM method implemented in the edgeR package (version 1.16.0) and expressed as CPM (counts per million). Bin-level coverage in each input sample was subtracted from that in the corresponding ChIP sample.
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| Submission date |
Jun 11, 2018 |
| Last update date |
Jun 07, 2024 |
| Contact name |
Ruben Esse |
| E-mail(s) |
ruben.esse@kcl.ac.uk
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| Organization name |
King’s College London
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| Department |
Department of Medical and Molecular Genetics
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| Street address |
Guy's Hospital, Great Maze Pond
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| City |
London |
| ZIP/Postal code |
SE1 9RT |
| Country |
United Kingdom |
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| Platform ID |
GPL19757 |
| Series (1) |
| GSE115551 |
Genome-wide mapping of H3K9me2 in DOT1L-deficient worms. |
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| Relations |
| BioSample |
SAMN09389330 |
| SRA |
SRX4190896 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3181878_dot1_K9me2_replicate_2.bedgraph.gz |
2.9 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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