|
| Status |
Public on Oct 26, 2012 |
| Title |
RNA-seq_HCT116_DKO |
| Sample type |
SRA |
| |
|
| Source name |
HCT116 with homozygous deletion of DNMT3b and DNMT1 (Rhee et al Nature 2002)
|
| Organism |
Homo sapiens |
| Characteristics |
technique: ds-cDNA-sequencing
starting molecule: RNA
antibody/capture: poly(A) selection (Oligotex mRNA Mini Kit, QIAGEN)
|
| Treatment protocol |
No specific treatments
|
| Growth protocol |
HCT116 WT and DKO cells were cultured in McCoy’s 5A medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (all Gibco/Invitrogen) at 37 °C in 5% CO2 atmosphere.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
100µg total RNA was subjected to two rounds of poly(A) selection (Oligotex mRNA Mini Kit, QIAGEN), followed by DNaseI treatment (QIAGEN). 100-200ng mRNA was fragmented by hydrolysis (5x fragmentation buffer: 200mM Tris acetate, pH8.2, 500mM potassium acetate and 150mM magnesium acetate) at 94°C for 90 s and purified (RNeasy MinElute Cleanup Kit, QIAGEN). cDNA was synthesized using 5µg random hexamers by Superscript III Reverse Transcriptase (Invitrogen). ds-cDNA synthesis was performed in second strand buffer (Invitrogen) according to the manufacturer's recommendations and purified (MinElute Reaction Cleanup Kit, QIAGEN).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Cluster generation and sequencing-by-synthesis (36 bp) was performed using the Illumina Genome Analyzer IIx (GAIIx) platform according to standard protocols of the manufacturer (Illumina). The image files generated by the Genome Analyzer were processed to extract DNA sequence data. Sequence reads were aligned to the human (hg18) reference genome using the Illumina Analysis Pipeline allowing, one mismatch. Only the 36 bp sequence reads uniquely aligning to the genome were considered for further analysis. The output data were converted to Browser Extensible Data (BED) files for downstream analysis and Wiggle (WIG) files for viewing the data in the UCSC Genome Browser. All sequence analyses were conducted based on the Homo Sapiens hg18 genome assembly.
Genome_build: hg18
Supplementary_files_format_and_content: The file "MergedPeaks_normalized_tagdensity.gz" contains processed data for all patient samples. It is a tab-delimited text file. MethylCap-seq was performed on 24 micro-dissected tumors and an equal number of matched microscopically normal tissue samples (48 DNA methylation profiles in total). Enriched regions (peaks) were called for each sample on the basis of a Poisson distribution of overlapping sequence reads within a dynamic window. A false discovery rate (FDR) was calculated relative to the total covered sequence, and peaks with an FDR of ≤10-6 were selected. All peaks from the 48 samples were merged. Per sample the number of tags overlapping each region of interest was calculated and normalized by dividing by the total number of aligned reads of the sample, and the length of the peak.
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| |
|
| Submission date |
Jul 03, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Arjen Brinkman |
| E-mail(s) |
arjen.brinkman@gmail.com
|
| Organization name |
Radboud University, Nijmegen Center for Molecular Life Sciences
|
| Department |
Molecular Biology
|
| Lab |
Stunnenberg
|
| Street address |
NCMLS #274, Geert Grooteplein Zuid 30
|
| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL10999 |
| Series (1) |
| GSE39068 |
Comparative genome-wide DNA methylation analysis of colorectal tumor and matched normal tissues |
|
| Relations |
| SRA |
SRX157407 |
| BioSample |
SAMN01085276 |
