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Sample GSM2058677 Query DataSets for GSM2058677
Status Public on Apr 12, 2016
Title HCT116kiTop1_Top1_Seq_mod
Sample type SRA
 
Source name HCT116 with homozygous Top1 knock-in
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
chip antibody: anti-TOP1 (ab109374)
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.
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.
Extracted molecule genomic DNA
Extraction protocol Cell were treated with CPT for 4 minutes and quickly incubated in TE-SDS 0.1%. Samples were sonicated to produce chromatin fragments of 300 bp on average. After clarification by centrifugation, sonicated extracts were adjusted to the conditions of FA_lysis buffer (50 mM HEPES, 150 mM NaCl, 2 mM EDTA pH 8.0 with 0.1% SDS). Standard ChIP was performed as previously described (Venters and Pugh, 2009).
Samples were immunoprecipitated as described for ChIP, treated with tyrosyl-DNA phosphodiesterase 1 (TDP1), poly-nucleotide kinase (PNK) and Illumina sequenced.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina Genome Analyzer II
 
Data processing library strategy: Top1-seq
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
 
Submission date Feb 09, 2016
Last update date May 15, 2019
Contact name Damian Wojtowicz
E-mail(s) wojtowda@mail.nih.gov
Phone 3014963713
Organization name NCBI/NLM/NIH
Street address 8600 Rockville Pike; Rm 8N811N
City Bethesda
State/province MD
ZIP/Postal code 20894
Country USA
 
Platform ID GPL9115
Series (1)
GSE57628 Study of Topoisomerase I in human
Relations
BioSample SAMN04482420
SRA SRX1569442

Supplementary file Size Download File type/resource
GSM2058677_HCT116_kiTop1_Top1_Seq_mod_ctrl_normalized.profile.wig.gz 595.7 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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