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| Status |
Public on Jul 06, 2016 |
| Title |
Thy_mm_RAG1_ChIPseq_rep1 |
| Sample type |
SRA |
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| Source name |
thymocytes
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| Organism |
Mus musculus |
| Characteristics |
cell type: thymocytes
genotype: wt
strain: C57BL/6
chip antibody: RAG1
age: 5 weeks
Sex: male
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For mouse thymocytes RAG1 ChIP-seq, cells were crosslinked with 1% HCHO, quenched with 0.125 M glycine, washed and resuspended in high-salt RIPA buffer (10 mM Tris pH 7.4, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.8 M NaCl, 1 mM PMSF, 1 μg/μL pepstatin A) and sonicated using a water bath sonicator (Diagenode) to obtain DNA of ≈ 100-250 bp.H3K27Ac ChIP-seq in REH cells was performed similarly, with the following modifications. After crosslinking, cells were resuspended in SDS Lysis Buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS, 1 mM PMSF, 1 μg/μL pepstatin A; 200 μls SDS Lysis Buffer per 10 million cells). For each immunoprecipitation, 300 μls of supernatant (corresponding to 15 million cells) was diluted to 1 mL in ChIP dilution buffer (167 mM NaCl, 16.7 mM Tris pH 8, 1.2mM EDTA, 1.1 % Triton X-100, 0.01% SDS) and pre-cleared with Protein G Dynabeads (Thermo-Fisher). The Dynabeads were then removed using a Dynal magnetic stand. After preclearing and adding blocking agent, anti-H3K27Ac antibody (5 μg; Abcam) was added, and the samples were incubated overnight at 4°C with rotation. Protein G Dynabeads were blocked with 2% BSA and 50 ng/μL heparin, and were then added to the samples, incubating for 2 hours at 4°C with rotation. The Dynabeads were washed (10 min, 4°C, with rotation, for each wash): 2x in Low Salt Wash Buffer (20mM Tris, pH 8, 150mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS and 1 mM DTT), 2x in High Salt Wash Buffer (20mM Tris, pH 8, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS and 1 mM DTT), 2x LiCl Wash Buffer (10mM Tris pH 8, 1mM EDTA, 0.25 M LiCl, 0.5% NP-40, 1% sodium deoxycholate), 1x in TE + 0.2% Triton X-100, and 1x in TE. Beads were incubated twice with 150 μls of elution buffer (1% SDS, 0.1 M NaHCO3) for 15 minutes at room temperature. Eluates were pooled and incubated at 65°C overnight followed by treatment with RNase and Proteinase K. DNA was purified by standard phenol:chloroform extraction and ethanol precipitation.
libraries were prepared and sequenced following standard Illumina protocols
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
control: Input and RAG1-/-
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| Data processing |
mouse and human ChIP-seq reads were aligned to the GRCm38p2/mm10 and GRCh37/hg19 genome assemblies,respectively.
Alignment done using bowtie version 0.12.7, with the parameters: --best --all --strata -n 2 -m1 -l 50
peaks were called using MACS version 1.4.2 with default parameters
Genome_build: GRCm38p2/mm10 for mouse and GRCh37/hg19 for human
Supplementary_files_format_and_content: wig files were generated using MACS with parameter --space 100, and then normalized to RPM. Scores in the wig files represent the averaged RPM over overlapping 100bp window
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| Submission date |
Jul 05, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Yaakov Maman |
| Organization name |
Bar-Ilan University
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| Department |
Azrieli Faculty of Medicine
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| Lab |
Genome Instability & Cancer
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| Street address |
Henrietta Szold 8
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| City |
Safed |
| ZIP/Postal code |
1311502 |
| Country |
Israel |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE84052 |
RAG1 targeting in the genome is dominated by chromatin interactions mediated by the non-core regions of RAG1 and RAG2 |
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| Relations |
| BioSample |
SAMN05360362 |
| SRA |
SRX1898035 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2226524_T_mm_R1_rep1.wig.gz |
7.3 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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