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| Status |
Public on Sep 02, 2015 |
| Title |
CD4SP_dKO |
| Sample type |
SRA |
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| Source name |
Thymus
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| Organism |
Mus musculus |
| Characteristics |
cell type: Mature CD4 SP Thymocytes
genotype: Kdm6bf/f, Kdm6af/f Cd4-Cre+
chip antibody: H3K27me3 (Millipore, 17-622)
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| Treatment protocol |
NA
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| Growth protocol |
NA
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Sorted CD69lo wt DP thymocytes and mature (TCRhi or Vα2hi CD24lo) CD4 SP thymocytes from Wt, Jmjd3KO, UtxKO and dKO mice were digested with MNase, followed by sonication to generate mononucleosomes. Sonicated supernatants were pre-cleared with protein G beads and then immunoprecipitated with anti-H3K27Me3.
The libraries were prepared according to standard Illumina's protocol. The ChIP DNA is blunt-ended and phosphorylated. A single 'A' nucleotide is added to the 3' ends of the fragments in preparation for ligation to an adapter that has a single-base 'T' overhang. The ligation products are purified and size-selected. The size-selected DNA is purified and PCR-amplified to enrich for fragments that have adapters on both ends. The final purified product is then quantitated by qPCR before cluster generation and sequencing. Clusters were generated on an Illumina flow cell and libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Data processing |
Illumina CASAVA_v1.7 was used for demultiplex and converting binary basecalls and qualities to qseq.txt format, and CASAVA_v1.8.2 was used for demultiplex and converting binary basecalls and qualities to fastq format.
The fastq files (36 bp, single end) were aligned against the genome reference mm9 and promoter reads (+/- 2Kb from the transcription start site) detected for each gene using the Rsubread package in R (1).
Promoter counts were normalized by loess using csaw package (2), and broad peaks were detected using SICER (3)
Promoter reads overlapping with SICER peaks in two biological replicates per group (KO and WT) were further analyzed for differential binding using the glmQLFit function in EdgeR (4,5).
Offsets were used to generate normalized bedgraph files for visualization in IGV.
Genome_build: mm9
Supplementary_files_format_and_content: bedgraph
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| Submission date |
Jul 10, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Margaret C Cam |
| E-mail(s) |
[email protected]
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| Phone |
240-760-7179
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| Organization name |
NIH
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| Street address |
Bldg 37/3041C
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL9185 |
| Series (1) |
| GSE70795 |
Impact of Jmjd3 and Utx histone demethylases on Histone H3 lysine 27 trimethylation (H3K27Me3) in mature CD4 SP thymocytes |
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| Relations |
| BioSample |
SAMN03854648 |
| SRA |
SRX1091914 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1818904_CD4SP_dKO.bedgraph.gz |
170.1 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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