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| Status |
Public on Apr 12, 2016 |
| Title |
HCT116kiTop1_BRD4_+JQ1_ChIPSeq |
| Sample type |
SRA |
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| Source name |
HCT116 with homozygous Top1 knock-in + JQ1
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
tissue source: Epithelial Tumor Colon
cell type: human colon cancer cells
genotype/variation: 46,XY,add(10)(q26),add(16)(p13.3),add(18)(p11.2)[2]; 45,idem,-Y[17] and 45,idem,-16[1]; and TOP1-KI
treated with: 250 nM (final con .) JQ1 for 2hrs
chip antibody: anti-BRD4 (A301-985A)
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| Treatment protocol |
CPT (Sigma) treatments were performed for 10 mins on exponentially growing cells at 37ºC with a final concentration of 10 μM. JQ1 treatments were performed for 2 h with a final concentration of 250 nM.
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| Growth protocol |
Human colon cancer cells HCT116 and the derived HCT116-siTOP1 and HCT116KI were grown in DMEM supplemented with 10% heat-inactivated FCS. CRISPR-Cas9 System for HCT116kiTop1: MIT CRISPR Design Tool selected small guide RNAs targeting TOP1 exon 4 that were cloned in pX330-PGK-puro-SV40PA (gift of R. Casellas; NIAID, NIH). The recombination donor was made by cloning a gBlock Gene Fragment in a ColE1 origin vector. The gBlock contained 3 HA tag sequences replacing most of exon 4, flanked by two homology arms. Following puromycin selection, clones were screened by genomic PCR, and verified by sequencing and immunoblot. Positive clones were grown from single cells prior to experiments.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP samples were prepared from HCT116, HCT116-siTOP1 and HCT116KI cells following Barski et al. protocol (Barski et al., 2007) with minor changes. Briefly, 5x107 cells were cross-linked with 1% formaldehyde for 10 min. Cross-linking was stopped by the addition of glycine to 125 mM final concentration and cells were washed twice with PBS. After harvesting cells by scraping, the pellet was washed once with PBS plus 0.5% BSA and resuspended in TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) supplemented with complete protease inhibitor tablet (Roche) to a final concentration of 1x106 cells/ml. Samples were sonicated 20 times with 20-sec pulses, 30 sec resting, using the Ultrasonic Processor XL (HEAT System) to produce chromatin fragments of 300 bp on average. After clarification by centrifugation, sonicated extracts were adjusted to the conditions of RIPA buffer by adding 1% Triton X100, 0.1% Na-Deoxycholate, 0.1% SDS and 200 mM NaCl. 4 μg of anti-RNAPII (ab817) or anti- TOP1 (ab109374) or anti-BRD4 (A301-985A) were mixed with 40 μl of Dynabeads Protein A (Invitrogen) and incubated at 4°C for 6 hr with rotation. Chromatin from 5×106 cells was added to the Protein A-antibody complexes and incubated overnight at 4°C with rotation. Immunoprecipitates were washed twice with RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 1% Triton X100, 0.1% Na-Deoxycholate, 0.1% SDS, 200 mM NaCl); twice with RIPA buffer plus 300 mM NaCl; twice with LiCl buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-Deoxycholate); and twice with TE. The beads were then resuspended in TE plus 0.25% SDS supplemented with proteinase K (500 μg/ml, Roche) and incubated overnight at 65ºC. After pooling, the DNA was recovered from the eluate by phenol chloroform extraction followed by ethanol precipitation in the presence of 20 μg of glycogen (Roche) and dissolved in TE.
The Epicentre DNA END-Repair kit (Epicentre Biotechnologies) was used to generate blunt-ended DNA. DNA was incubated for 45 min at room temperature with a mixture of End repair buffer (33 mM Tris-acetate pH 7, 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DTT), 0.25 mM of each dNTPs, 1 mM ATP, and 1 μl End-Repair Enzyme mix (T4 DNA polymerase + T4 PNK). After purification, the blunt-ended DNA was treated with 15 units of Klenow(exo-) for 30 min at 37°C in the presence of 0.2 mM dATP to generate a protruding 3′ A base used for adaptor ligation. Illumina adapter was ligated to the end of DNA fragments by incubating with 0.1 μl Adaptor oligo mix and 1,000 units of T4 DNA ligase at room temperature for 30 min. After one step of DNA purification using QIAquick PCR Purification Kit the DNA was eluted. A size selection of the adapter ligated DNA was performed through 2% E-Gel (Invitrogen) electrophoresis. The gel slice, around the 200-400 bp region was excised, then DNA was extracted using the MinElute gel extraction kit (QIAGEN) in a final volume of 12 μl elution buffer. The DNA was then amplified for 18 cycles using Illumina primers (Fw: 5'-aca ctc ttt ccc tac acg acg c-3'/ Rv: 5'-caa gca gaa gac ggc ata cga gc-3') according to the following protocol: 98°C for 30 sec; 98°C for 10 sec; 65°C for 30 sec; 72°Cfor 30 sec. The PCR product was obtained by excising 220bps-500bps DNA from a 2.5% agarose gel and purifying it through a Qiagen gel extraction kit (QIAGEN).The purified DNA was used directly for cluster generation and sequencing analysis using the Illumina Genome Analyzer following manufacturer protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
Illumina Analysis Pipeline
PolII ChIP-Seq, BRD4 ChIP-Seq, Top1 ChIP-Seq and Top1-Seq sequencing reads were aligned to the hg19 genome assembly using Bowtie 2 version 2.2.2. To minimize potential PCR bias, duplicate reads were removed from ChIP-Seq data using SAMtools. Peaks were called in RNAPII and Top1 ChIP-Seq data using QuEST software (version 2.4) with a random background model; signal enrichment in Top1 ChIP-Seq data was sought in wide regions, as Top1 did not have a narrow binding sites. All reads of Top1-Seq sample are presented in 50bp bins for each replicate separately.
Genome_build: hg19
Supplementary_files_format_and_content: ChIP-Seq peaks, RNA-seq abundance measurements
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| Submission date |
Feb 09, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Damian Wojtowicz |
| E-mail(s) |
wojtowda@mail.nih.gov
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| Phone |
3014963713
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| Organization name |
NCBI/NLM/NIH
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| Street address |
8600 Rockville Pike; Rm 8N811N
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20894 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (1) |
| GSE57628 |
Study of Topoisomerase I in human |
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| Relations |
| BioSample |
SAMN04482417 |
| SRA |
SRX1569439 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2058674_HCT116_kiTop1_BRD4_ChIPSeq_JQ1_normalized.profile.wig.gz |
327.5 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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