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Sample GSM1385719 Query DataSets for GSM1385719
Status Public on Apr 12, 2016
Title HCT116_wt_RNASeq
Sample type SRA
 
Source name human colon cancer cells HCT116
Organism Homo sapiens
Characteristics cell line: HCT116
cell type: Epithelial Tumor Colon
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]
treatment: untreated
chip antibody: none
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.^SAMPLE = GSM1385718
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.^SAMPLE = GSM1385719
Extracted molecule total RNA
Extraction protocol Total RNAs were purified and quality check was performed on Agilent Bioanalyzer. RNA-Seq library preparation and sequencing for HCT116 cells followed the procedure described in Chepelev, I., et al., Detection of single nucleotide variations in expressed exons of the human genome using RNA-Seq. Nucleic Acids Res, 2009. 37(16): p. e106.
As described in Chepelev, I., et al., Detection of single nucleotide variations in expressed exons of the human genome using RNA-Seq. Nucleic Acids Res, 2009. 37(16): p. e106.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina Genome Analyzer II
 
Data processing Illumina/ABSOLiD 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.
RNA-Seq short reads were aligned against the human (hg19) genome using Tophat (version 1.4.1) with default settings. Resulting alignments were analyzed using Cufflinks software (version 1.3.0) provided with the annotation of Ensembl 60 genes after removing germline antigen fragments, mitochondrial rRNA genes, miRNAs and small nuclear RNAs. Expression values were determined in terms of Fragments Per Kilobase of exon per Million fragments mapped (FPKM).
Genome_build: hg19
Supplementary_files_format_and_content: Files report ChIP-Seq peaks, RNA-seq abundance measurements
 
Submission date May 13, 2014
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
SRA SRX534907
BioSample SAMN02767061

Supplementary file Size Download File type/resource
GSM1385719_genes.fpkm_tracking.txt.gz 1.2 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data included within Sample table
Processed data provided as supplementary file

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