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| Status |
Public on Sep 13, 2013 |
| Title |
ChIP Stat5a replicate |
| Sample type |
SRA |
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| Source name |
purified memory TCRP1A eTC-STAT5CA
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| Organism |
Mus musculus |
| Characteristics |
cell type: purified memory TCRP1A eTC-STAT5CA
tissue: Blood
passages: N.A.
strain: C57BL/6
developmental stage: memory CD8+ T cells
chip antibody: anti-Stat5a
chip antibody manufacturer: R&D Systems
chip antibody catalog #: PA-ST5A
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| Treatment protocol |
CD8 T cells were prepared from LN or spleen according to the standard procedures. Cell suspensions were passed over Ficoll-PaqueTM solution (Amersham Biosciences AB).
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| Growth protocol |
TCRP1A CD8 T lymphocytes were activated by their cognate P1A Ag. After 24h, an active form of Stat5 (STAT5CA) was introduced in activated cells. Culture was continued for another 48h to induce their differentiation in effector T cells. These 72h activated T cells were injected in congeneic hosts and recovered 70 days later from hosts’ spleen and lymph nodes: TCRP1A eTC-STAT5CA.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin Preparation (ChIP): EDTA-free protease inhibitors (Roche, France) were added to all washing buffers to a finalconcentration of 1X. Aliquots of 5x107 cells were lysed after resuspension in 2 ml LB1 (50mM Hepes pH 7.5, 140mM NaCl, 1mM EDTA pH 8, 10% glycerol, 0.75% NP-40, 0.25% Triton X-100) for 20 minutes on a rotating wheel at 4°C. Chromatin was collected by centrifugation for 5 minutes at 4°C and 1.350g in a tabletop centrifuge, and washed in 2 ml LB2 (200mM NaCl, 1mM EDTA pH 8, 0.5mM EGTA pH 8,10mM Tris pH 8) on a rotating wheel for 10 minutes at 4°C. Chromatin was collected,resuspended in 2 ml of LB3 (1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 100mMNaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) and sonicated using a Misonix 4000(Misonix Inc, USA) sonicator for 16 cycles (30 seconds on, 30 seconds off, amplitude 40) with the tubessubmerged in ice-cold sonication solution (500mM NaCl, 20% ethanol). After the addition ofTriton X-100 to a final concentration of 1%, particulates were collected through centrifugation at20.000g and 4°C for 20 minutes. For batch processing, supernatants were combined, mixedthoroughly and subsequently aliquoted and snap-frozen in liquid nitrogen. Extracts were kept at-80°C until use and 50μl aliquots were taken to serve as input controls. Inputs were combined with an equal volume of 2x elution buffer (100mM Tris pH 8, 20mM EDTA pH 8, 2% SDS) and incubated overnight in a water bath at 65°C for 13-15 hours. SDS was then diluted by the addition of an equal volume of TE (10mM Tris pH 8, 1mM EDTA pH 8) and RNA was digested by RNase A at a final concentration of 0.2μg/ml at 37°C for 2 hours. Samples were subsequently Proteinase K treated at 55°C for two hours at a final concentration of0.2μg/ml. DNA was purified by two subsequent phenol:chloroform:isoamylalcohol (25:24:1, pH8) extractions and followed by a Qiaquick purification (PCR purification columns, Qiagen,Germany). To ensure greater DNA purity, columns were washed twice with PE buffer and elutedin 50μl H2O. DNA concentration was measured using a Nanodrop 1000 (Thermo Scientific,France) and 1 ul was used to verify sonication efficiencies using High Sensitivity DNA chips on a 2100 Bioanalyzer. ChIP experiments were performed using Dynabeads (Invitrogen, USA) coated with Protein-G. Beads were washed 3x with 1ml and subsequently resuspended in 250μl of blocking solution (0.5% BSA in 1x DPBS). After the addition of the antibody (anti-Stat5a; PA-ST5A from R&D Sysytems), the beads were incubated at 4°C overnight on a rotating wheel. Unbound antibodies were removed through three further washes with 1ml of blocking solution. Beads were resuspended in 100μl of blocking solution, extracts were added and the mix was incubated overnight at 4°C on a rotating wheel. EDTA-free protease inhibitors (Roche) were added to all washing buffers to a final concentration of 1x. Beads were washed 8 times in RIPA buffer (50mM Hepes pH 7.6, 500mM LiCl, 1mM EDTA pH 8, 1% NP-40, 0.7% Na-Deoxycholate) and once in TE+ (10mM Tris pH 8, 1mM EDTA pH 8, 50mM NaCl). Immunoprecipitated chromatin was recovered from the beads with two subsequent elution steps at 65°C for 15 and 10 minutes in 60μl and 40μl of elution buffer (50mM Tris pH 8, 10mM EDTA pH 8, 1% SDS) respectively. The two eluates were combined and incubated at 65°C overnight (13-15 hours) for crosslink reversal. DNA was purified as described for the input. Sequencing 10 ng of Input and 90 µl of STAT5a ChIP were used for high throughput sequencing. Standard Applied Biosystems Life Technologies protocols were followed for the creation of short-insert bar-coded libraries (insert length ~150 bp) according to the ChIP-seq Kit Guide from the manufacturer (cms_079323.pdf) and library preparation guide (cms_081748.pdf). The library was PCR amplified with 15 cycles. The amplified fragments were verified on a 2100 Bioanalyzer (Agilent, USA) before sequencing on an AB SOLiDTM V4.0 or hq5500XL (Life Technologies) according to the manufacturer’s protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB 5500xl Genetic Analyzer |
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| Description |
ChIP
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| Data processing |
Pre-processing Sequence reads of 50 and 75 bp were produced respectively with a SOLiDTM V4.0 or hq5500xl sequencers, according to standard Life Technologies protocols. Sequence read mapping to version 9 build 37 (mm9) of the mouse genome was achieved with Bfast (v0.7.0), only reads with a unique best scoring alignment and mapping once to the genome were retained for subsequent analyses, to prevent spurious calls in repetitive regions. To reduce the risk of potential bias, we discarded all tags with exactly the same coordinates. Generation of Wigged files Reads were elongated to 204 bp and 189 bp for replicates 1 and 2, respectively. The values were summed up in non-overlapping 50 bp bins along each chromosome to generate Wigged files using a R custom script. Peak calling Significant STAT5 bound regions were retrieved using the HOMER peak-calling tool (http://biowhat.ucsd.edu/homer/) with a FDR of 0.001, and a local input fold enrichment of 4.
genome build: MM9
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| Submission date |
Jun 25, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Nathalie AUPHAN-ANEZIN |
| E-mail(s) |
auphan@ciml.univ-mrs.fr
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| Phone |
+33 491 269 189
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| Organization name |
CIML
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| Street address |
Parc Scientifique de Luminy
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| City |
MARSEILLE |
| ZIP/Postal code |
13009 |
| Country |
France |
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| Platform ID |
GPL15907 |
| Series (1) |
| GSE41818 |
Active STAT5 in CD8 T cells imprints a T-Bet-dependent Tc-1 program with repressed IL-6/ TGFb1 signaling leading to enhanced anti-tumor responses |
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| Relations |
| BioSample |
SAMN02213868 |
| SRA |
SRX315127 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1173872_Tcd8_STAT5_2_TAGC.hmr.bed.gz |
178.8 Kb |
(ftp)(http) |
BED |
| GSM1173872_Tcd8_STAT5_2_TAGC.wigNorm.wig.gz |
9.8 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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