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| Status |
Public on Jun 15, 2015 |
| Title |
Pol II_2_GFP_ChIPSeq |
| Sample type |
SRA |
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| Source name |
Jurkat T cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: T Cells
cell line: Jurkat
transgene: GFP
chip antibody: Pol II CTD 4F8(Active motif; 61081)
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| Treatment protocol |
To induce expression of GFP:SF and Tat:SF for RNA-Seq and ChIP-Seq the stable cell lines were treated with 1 μg/ml doxycycline for 16 hrs.
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| Growth protocol |
CD4+ Jurkat T cells (clone E6.1) were cultured in RPMI1640 (HyClone) with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen). Jurkat T-Rex cells were maintained in the same conditions but in the presence of 10 mg/ml Blasticidin as indicated by the manufacturer's instructions (Invitrogen). The derived Jurkat T-Rex clones (GFP bearing the Strep/Flag (SF) epitope and Tat bearing the SF epitope) were selected with 300 mg/ml of zeocin for four weeks and later cultured with 10 mg/ml Blasticidin and 100 mg/ml zeocin (Invitrogen). To generate the stable GFP:SF and Tat:SF expressing cell lines, the parental Jurkat T-Rex was electroporated with 2 mg of Ssp1-linearized pcDNA4\TO vector (Invitrogen) bearing either insert using a nucleofector kit V and a Nucleofector II (Lonza).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was cross-linked with 1% formaldehyde for 10 min at room temperature in culture media, and the reaction was stopped by the addition of glycine (125 mM final). Cells were washed twice with PBS and re-suspended in 1 ml of nuclei extraction buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1 mM PMSF, protease inhibitor (Roche)) at 2x107 cells/ml and incubated for 10 min at 4°C. Cell nuclei were collected by centrifugation at 3000 rpm for 5 min at 4°C, and re-suspended in Szak's RIPA buffer (50 mM Tris-HCl pH 8.0, 1% NP-40, 150 mM NaCl, 0.5% deoxycholate, 0.1% SDS, 5 mM EDTA, 0.5 mM PMSF, protease inhibitor) at 5x107 nuclei/ml. Nuclear pellets were sonicated using the Bioruptor water bath sonicator (Diagenode) using the high setting with 20 sec on 30 sec off for 45 min to generate 100-300 bp DNA fragments. Chromatin DNA was quantified with NanoDrop 1000 (Agilent). For the ChIP step, 1–3 mg of antibody was conjugated with 15 ml of protein G dynabeads (Invitrogen) and blocked with 0.16% bovine serum albumin for 16 hrs at 4°C. Antibody-conjugated dynabeads were incubated with 30–50 μg of chromatin DNA for 2 hr at 4°C. Then the beads were sequentially washed sequentially two times with the following buffers: low salt buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton, 0.1% SDS, 2 mM EDTA), high salt buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 1% Triton, 0.1% SDS, 2 mM EDTA), LiCl wash buffer (0.25M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 20 mM Tris, pH 8.0), and 1 mM Tris-EDTA pH 8.0. Chromatin immunocomplexes were eluted by incubation for 10 min at 65°C with 1% SDS and 100 mM NaHCO3, and cross-linking was reversed by incubation in the solution adjusted to 200 mM NaCl and proteinase K (20 μg) for 1 hr at 65°C.
ChIP DNA was submitted to McDermott Center Sequencing Core at UT Southwestern Medical Center for library preparation and high-throughput sequencing. ChIP DNA was quantified on a Qubit® 2.0 Fluorometer (Invitrogen) and libraries from ~10 ng ChIP DNA were prepared using the SOLiDTM ChIP-Seq Kit with modifications for the 5500xl instrument (Applied Biosystems). Samples were end repaired, 3’-end adenylated and barcoded with multiplex adapters (Applied Biosystems). After purification with Ampure XP beads (Beckton Coulter), samples were PCR amplified (~14 cycles), size selected with Ampure XP beads, and quantified on the Agilent 2100 Bioanalyzer. Emulsion PCR was then performed and beads enriched on the EZ-Bead system.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB 5500xl Genetic Analyzer |
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| Data processing |
ChIP-Seq reads were aligned to the reference genome hg19 using bowtie v1.1. The flag "-m 1" was used
Peaks were called using macs2 using the default fdr threshold of p-value < 0.05
Genome_build: hg19
Supplementary_files_format_and_content: ChIP-Seq: Tab-delimited peak files generated by macs2
Note: When converting SRA files to FASTQ files using fastq-dump (tested with sra toolkit v2.8.0), please refer to submitter recommendations in README file linked as supplementary file on GSE65687 record.
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| Submission date |
Feb 05, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Ivan D'Orso |
| E-mail(s) |
ivan.dorso@utsouthwestern.edu
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| Phone |
214-633-1374
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| Organization name |
UT Southwestern Medical Center
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| Department |
Microbiology
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| Lab |
NL3.110A
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| Street address |
5323 Harry Hines
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| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390-9048 |
| Country |
USA |
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| Platform ID |
GPL16288 |
| Series (2) |
| GSE65687 |
Tat controls RNA Polymerase II and the epigenetic landscape to precisely rewire cellular transcriptional programs (ChIP-Seq) |
| GSE65689 |
Tat controls RNA Polymerase II and the epigenetic landscape to precisely rewire cellular transcriptional programs |
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| Relations |
| BioSample |
SAMN03331660 |
| SRA |
SRX867191 |
| Named Annotation |
GSM1603219_GFP_4F8_treat_pileup.bw |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1603219_GFP_4F8_treat_pileup.bw |
86.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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