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| Status |
Public on Jun 09, 2016 |
| Title |
NELF-EKD_VEH_GFP_IgG_2 |
| Sample type |
SRA |
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| Source name |
NELF-E Knockdown, vehicle, GFP, IgG
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| Organism |
Homo sapiens |
| Characteristics |
cell type: human breast cancer cells
cell line: MCF-7
chip antibody: IgG
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| Treatment protocol |
MCF-7 Cells were grown in (1) MEM supplemented 1 µg/mL doxycycline for four all four days, (2) MEM with charcoal stripped calf serum for three days, and (3) vehicle (DMSO) or DRB (100 µM) containing medium (1:1000 dilution of DMSO) for 1 hour prior to their crosslinking with 1% formaldehyde in PBS. Crosslinking was quenched with 0.1 M Glycine after 10 minutes at room temperature, and cells were harvested with cold PBS for sonication (200-500 bp) and chromatin immunoprecipitation. MEFs were harvested in cold PBS and collected by centrifugation. The cells were swollen in Nuclei Isolation Buffer (10 mM HEPES, pH 8.0, 2 mM MgCl2, 3 mM CaCl2, 300 mM sucrose, with freshly added 1 mM DTT, protease inhibitors, and phosphatase inhibitors) and the nuclei were released by the addition of 0.65% NP-40 with moderate vortexing. Following collection by centrifugation, the nuclei were resuspended in PARP Reaction Buffer (30 mM Tris•HCl, pH 7.5, 10 mM KCl, 5 mM MgCl2, 5 mM CaCl2, 0.01% NP-40, 0.05 mM EDTA, 20% glycerol, with freshly added 1 mM DTT, protease inhibitors, and phosphatase inhibitors) containing 250 μM 8-Bu(3-yne)T-NAD+ for 30 minutes at 25°C with occasional gentle mixing to allow ADP-ribosylation to occur in the isolated nuclei.
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| Growth protocol |
MCF-7 cells were grown in culture as previously described (Hah et al. Cell 2011). PARP-1 knockout MEFs expressing analog-senstive PARP-1 were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin, and streptomycin containing 1 µg/mL puromycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
PARP-1 ChIP-seq: Following mock ADP-ribosylation in intact nuclei from MEFs as described above, the nuclei were crosslinked with 0.5% formaldehyde and clicked to biotin. Nuclei were sonicated in a water bath sonicator to a DNA fragment size distribution of 200 to 500 bp and chromatin was used for chromatin immunoprecipitation. Click-ChIP-seq: Following 8-Bu(3-yne)T-ADP-ribosylation in intact nuclei from MEFs as described above, the nuclei were crosslinked with 0.5% formaldehyde and clicked to biotin. Nuclei were sonicated in a water bath sonicator to a DNA fragment size distribution of 200 to 500 bp and bound to streptavidin-conjugated magnetic beads. Following extensive washing, the beads were collected in a magnetic field and de-crosslinked overnight at 65°C. DNA was to be sequenced was recovered by magnetic separation from the beads and purified by PCIAA extraction and ethanol precipitation. All Other ChIP-seq: Cells were sonicated in a water bath sonicator to a DNA fragment size distribution of 200 to 500 bp and chromatin was used for chromatin immunoprecipitation.
After purification, 50 ng of ChIP’d DNA for each condition was used to generate libraries for sequencing, as previously described {Robertson, 2007 #41}, with some modifications. Briefly, the DNA was end-repaired and a single “A”-base overhang was added using the Klenow fragment of E. coli DNA polymerase. The A-modified DNA was ligated with Ilumina sequencing adaptors using the Illumina TruSeq DNA Sample Prep Kit. The ligated DNA (250 ± 25 bp) was size-selected by agarose gel electrophoresis and extraction, amplified by PCR, and purified using AmPure beads (Beckman Coulter). The final libraries were subjected to QC (size, purity, adapter contamination) and sequenced using an Illumina HiSeq 2000 per the manufacturer’s instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
The ChIP-seq reads were aligned to the mm9 or hg19 human reference genome using the Bowtie software package (Langmead et al, 2009) .
Mapped reads were further converted to “bed” files for all analyses
Genome_build: mm9 or hg19
Supplementary_files_format_and_content: bed
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| Submission date |
May 12, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
W. Lee Kraus |
| E-mail(s) |
lee.kraus@utsouthwestern.edu
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| Organization name |
UT Southwestern Medical Center
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| Street address |
5323 Harry Hines Blvd.
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| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390-8511 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE74141 |
NAD+ Analog-sensitive PARPs Reveal a Role for PARP-1 in Transcription Elongation |
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| Relations |
| BioSample |
SAMN04994392 |
| SRA |
SRX1758514 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2151665_NELF-EKD_VEH_GFP_IgG_2.bed.gz |
275.0 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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