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| Status |
Public on Apr 20, 2010 |
| Title |
Thymocytes_R2-/-beta_RAG2 |
| Sample type |
SRA |
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| Source name |
Total thymocytes
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| Organism |
Mus musculus |
| Characteristics |
genotype: R2-/-b
biomaterial_type: primary cells
cell type: total thymocytes
chip antibody: RAG2 (rabbit polyclonal antibody raised to RAG2 aa 70-516 (Leu and Schatz, PMID: 7565717))
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Flow cytometry: Developing lymphocytes were analyzed or purified by flow cytometry using a Becton-Dickinson FACS Caliber or FACS Aria. Cells were stained with the following antibodies from BD Biosciences: anti-mouse CD4-PE (#553653), anti-mouse CD8-PE-Cy5 (#553034). Thymocytes were almost entirely CD4+CD8+ pre-T cells.
Chromatin immunoprecipitation - A) Crosslinking and sonication of chromatin: Thirty million cells were resuspended in 9 ml RPMI/2% fetal bovine serum in a 15 ml conical tube and 0.25 ml of 37% formaldehyde (J. B. Baker #2106-10) were added and the tube rocked for 15 min at room temperature (RT). Glycine (American Bioanalytical #56-40-6) was added to a final concentration of 0.125 M using a 1.25 M stock solution (pH 7.4) to terminate crosslinking. Cells were pelleted (3K, 3 min, 4°C) and washed twice with 10 ml cold PBS. Wash solutions contained 1 mM PMSF and 1 ug/ul pepstatin A, which were included in all subsequent steps. After the second wash, cells were resuspended in 1 ml PBS and transferred to a 1.5 ml microfuge tube, pelleted (3K, 3 min, 4°C), and the supernatant removed. Unless used immediately, cell pellets were frozen on dry ice and stored at -80°C. A cell pellet was resuspended in 600 ul RIPA buffer (10 mM Tris pH 7.4, 1 mM EDTA, 1% TritonX100, 0.1% sodium deoxycholate, 0.1% SDS) containing 0.8 M NaCl and incubated for 10 min on ice. Chromatin was sheared using a water bath sonicator (Diagenode) (4 x 5 min, high power) resulting in DNA of 300 to 500 bp. Samples were centrifuged (14K, 10 min, 4°C) and the supernatant transferred to a new microfuge tube. If not used immediately, 0.1 vol 50% glycerol was added and the sheared chromatin was stored at -80°C. Chromatin derived from 3-5 million cells was used for one ChIP experiment (one IP with a single antibody).
B) Pre-clearing of chromatin with Protein G beads: To create sufficient Protein G beads loaded with rabbit Ig to pre-clear the chromatin from 20 million cells, the following were mixed: 25 ul (bed volume) of magnetic Protein G agarose beads (Invitrogen #100-03D), 40 ul normal rabbit serum (Invitrogen, #16120-099), and 40 ul PBS. The mixture was rocked for 15 min at RT, spun (3K, 1 min), the pellet washed twice with 1 ml RIPA (0.8 M NaCl), and the buffer removed. A 10X volume of RIPA (0.8 M NaCl) was added to sheared chromatin, and the chromatin was then added to the rabbit Ig-loaded beads and rocked 1 hr at 4°C. Beads sedimented and the supernatant transferred to a new tube which contained a second batch of rabbit Ig-loaded beads, and rocked 1 hr at 4°C. Beads were sedimented again and the supernatant transferred to new tube. The precleared chromatin was stored at -80°C in aliquots if not used immediately.
C) Immunoprecipitation: Precleared chromatin was mixed with 2% fish skin gelatin (Sigma G7756) and 0.5 mg/ml heparin (Sigma H6279), BSA (final concentration 0.5 ug/ul), and 5 ug anti-RAG1, anti-RAG2 or anti-H3K4me3 and rocked at 4°C overnight. The binding reaction was centrifuged to remove any particulate matter (14K, 10 min, 4°C) and the supernatant transferred to a new tube containing 5 ul (bed volume) protein G beads (preblocked with 2% BSA for 1 hr at RT). This mixture was rocked for 3 hr at 4°C, beads were sedimented magnetically, and washed three times (10 min, 4°C) with 1 ml RIPA (0.8 M NaCl) containing 1 mM DDT, 100 mM PMSF, then once (10 min, 4°C) with 1 ml RIPA (0.3 M NaCl) containing 1 mM DDT, 100 mM PMSF, then once (10 min, 4°C) with 1 ml RIPA containing 1 mM DDT, 100 mM PMSF, and finally once (10 min, 4°C) with 1 ml TE (10 mM Tris-HCl (pH 7.4), 1 mM EDTA). Beads were resuspended in 100 ul TE containing 50 ug Proteinase K and 0.25% SDS, and incubated at 60°C overnight. Proteinase K digests were extracted with 100 ul phenol/chloroform and after transferring the aqueous phase to a new tube, the organic phase was back extracted with 100 ul of water, and the aqueous phases were pooled. The aqueous material was extracted with 200 ul chloroform and after transferring the aqueous phase to a new tube, the organic phase was back extracted with 200 ul of water, and the aqueous phases were pooled. NaCl was added to a final concentration of 0.2 M and DNA precipitated with 20 ug glycogen and 1 ml ethanol. The DNA pellet was washed with 70% ethanol and resuspended in 50 ul water.
D) Library preparation: For the RAG2 antibody, three immunoprecipitations (each using chromatin from 30 x 106 cells) were pooled to obtain sufficient DNA for sequencing. The ChIP DNA ends were repaired using PNK and Klenow enzymes, followed by treatment with Taq polymerase to generate a protruding 3? A base used for Illumina adaptor ligation. ChIP DNA was then amplified using adaptor primers for 17 cycles and fragments of approximately 200 bp (mononucleosome + adaptors) were isolated using an agarose gel. The purified DNA was used directly for cluster generation and sequencing analysis.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Rag2-/- 2B4 Tcrb transgenic thymocytes; RAG2 IP
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| Data processing |
Image analysis, base calling, quality filtering: Standard settings of GAPipeline 1.3+ using PhiX control; Fastq files contain full length reads (36nts) that passed the chastity filter. Qualities are phred64.
Alignment and peak finder: Nucleotides 1-35 of reads were aligned with Bowtie 0.9.9.3 against mm9, allowing 1 mismatch. Reads that aligned uniquely were then filtered to remove redundancy and SICER 1.0.3 was used to identify regions with higher read density than expected from a random background model. A bedGraph-formated file of the peaks was created by normalizing density to 1M aligned reads.
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| Submission date |
Apr 05, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Seolkyoung Jung |
| Organization name |
NIH
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| Department |
NIAMS
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| Lab |
biodata mining and discovery section
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| Street address |
10 Center Dr
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| City |
bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL9250 |
| Series (1) |
| GSE21207 |
The In Vivo Pattern of Binding of RAG1 and RAG2 to Antigen Receptor Loci |
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| Relations |
| SRA |
SRX018806 |
| BioSample |
SAMN00010945 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM530315_1_thymocytes_R2mmbeta_RAG2_peaks.bedgraph.gz |
12.5 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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