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Sample GSM953280

Query DataSets for GSM953280

Status

Public on Apr 03, 2013

Title

RNA_Ad5 (RL-7)

Sample type

SRA

Source name

intestine, ileum, adenoma, Apc-Min/+

Organism

Mus musculus

Characteristics

strain: C57BL/6J
genotype: Apc-Min/+
tissue: intestine, ileum, adenoma
Sex: male
age: 15-19 weeks
animal id: 3584, MT-0500-R

Extracted molecule

total RNA

Extraction protocol

Intestinal samples were excised, transferred to buffer RLT/1mM DTT (Qiagen, Hilden, Germany) and homogenized in a Tissue Lyser (Qiagen) for 2 x 2 min, frequency set to 20 with a steel bead of 5 mm diameter (Qiagen). Samples were snap frozen and stored at -80°C until extraction of DNA and RNA using the Allprep DNA/RNA Mini kit (Qiagen). A DNase digest of the RNA was performed on the columns according to the manufacturer's instructions. RNA-seq and MeDIP-seq libraries were generated from the same samples.
RNA-Seq: 4 µg of total RNA was depleted for ribosomal RNA using the RiboMinus Euaryote Kit for RNA-seq (Invitrogen, #A1083708) following the manufacturer's instructions. The RiboMinus depleted RNA was then used for the generation of single end RNA-seq libraries using the Illumina sample prep kit, oligo only (#FC-102-1004) and a strand-specific protocol as described previously (Parkhomchouk et al., 2009, Nucleic Acids Res 37: e123. doi:10.1093/nar/gkp596). The generated cDNA libraries were size selected on a 2% agarose gel, and fragments of 150-200 nt were excised (corresponding to insert sizes of 80-130 nt) and purified using the QIAquick Gel extraction kit (Qiagen). Libraries were subjected to single-end sequencing.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer IIx

Description

Processed data files for this Sample: RNA_MeDIP_promoter_genebody_v2.txt

Data processing

Illumina 1.5 and 1.6 pipelines.
36mer RNA-seq reads were aligned to the mouse genome using Bowtie (version 0.12.5 – parameter set: -n 2 -l 36 -y --chunkmbs 256 --best --strata -k 1 -m 1) against the genomic reference UCSC mm9. Subsequently, reads that did not map to the genome were aligned to the cDNA reference ENSEMBL v58 in order to map reads spanning exon junctions. Then, uniquely mapped reads aligning to the sense strand of a gene were counted.
Annotations using ENSEMBL58.
Genome_build: mm9
Supplementary_files_format_and_content: Tab-delimited text files. File "RNA_MeDIP_promoter_genebody_v2.txt" contains the RNA-seq raw read counts per Ensembl gene (Version58) and the normalized read counts (rpm, reads per million: number of exon read counts per gene / sum of the exon read counts of the sample x 1 000 000); the raw MeDIP-seq read counts per sample for individual promoter regions (-1 kb to +0.5 kb) and for individual genes (sum of the reads mapping to a gene) and the relative read counts that were calculated relative to the sample with the highest number of reads. In addition, the file contains the results of the edgeR analysis for the RNA-seq and the MeDIP-seq data. For the MeDIP-seq data, also a ratio and a t-test as well as a score (-log10(p.value)*10)*log(ratio) are given for the comparison of the normal intestinal samples (B,N) vs. the adenoma samples. Files "hyper_500bp_Ad_vs_BN_v1.txt", "hypo_500bp_Ad_vs_BN_v1.txt", "hypo_DMR_merged_Ad_vs_BN_v1.txt", "hyper_DMR_merged_Ad_vs_BN_v1.txt" contain the identified differentially methylated regions and the rpm values of the MeDIP-seq data. In brief, mean rpm values were calculated for genome-wide 500 bp windows overlapping by 250 bp using MEDIPS. Subsequently, for each 500 bp window, we applied a Wilcoxon's test in order to assess significance of methylation differences between the 5 ApcMin/+ adenomas (Ad) versus the 3 ApcMin/+ normal intestine (N), 3 Apc+/+ wildtype normal intestine (B). Differentially methylated regions (cDMRs) were identified by filtering for 500 bp windows associated with Wilcoxon p-values < 0.01, a ratio of >1.33 or <0.75 and a minimum average signal of 0.25 rpm for one of the two groups (results for the 500 bp bins in: "hyper_500bp_Ad_vs_BN_v1.txt", "hypo_500bp_Ad_vs_BN_v1.txt"). Overlapping significant 500 bp windows were merged if their methylation status indicated an extending DMR (Results for the merged regions in files "hypo_DMR_merged_Ad_vs_BN_v1.txt", "hyper_DMR_merged_Ad_vs_BN_v1.txt").

Submission date

Jun 27, 2012

Last update date

May 15, 2019

Contact name

Lukas Chavez

E-mail(s)

l.chavez@dkfz.de

Organization name

German Cancer Research Center (DKFZ)

Street address

Im Neuenheimer Feld 280