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Sample GSM1116239 Query DataSets for GSM1116239
Status Public on Apr 10, 2013
Title WT_Ago2CLIP_Tagged
Sample type SRA
Source name Dicer f/f murine MSC TT-Flag-HA-hAgo2 cell line
Organism Mus musculus
Characteristics cell type: murine MSC cell line
genotype: Dicer f/f
strain: C57BL/6
rna type: Rnase I digested immunopurified RNA
Treatment protocol Tagged or Untagged hAgo2 expression was induced by doxycycline 48 hours prior to experiment, with 0.1 ug/ml dox at 0 hours and 1 ug/ml dox at 24 hours.
Growth protocol Monoclonal immortalized Dicer WT MSCs were cultured in Alpha-MEM supplemented with pen/strep and 10% fetal bovine serum.
Extracted molecule total RNA
Extraction protocol Sequencing was carried out on RNA fragments crosslinked to immunopurified hAgo2 that had undergone DNase digestion and limited RNase I digestion in lysate.
CLIP-seq RNA libraries were prepared for sequencing using a circulariaztion approach similar to Konig et al., 2011.
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2000
Description RNA fragments immunoprecipitated by tandem Flag/HA IP after UV crosslinking from immortalized clonal lines of Dicerf/f (Dicer WT) mesenchymal stem cells (MSCs) expressing a FLAG-HA-tagged hAgo2 construct were cloned and sequenced.
Data processing Library strategy: CLIP-Seq
Sequenced reads were trimmed for adapter sequence and mapped to the UCSC mouse genome build mm9 using Bowtie1. Reads mapping to >1 location or with >2 mismatches were discarded. Reads were collapsed based on identical 5? and 3? end mapped coordinates. To include reads mapping across exon-exon junctions, we extracted splice junction coordinates from the isoforms produced by Tophat from RNA-seq data and used custom python scripts to generate a fasta file of the sequences +/- 45 nt of these splice junctions. We then mapped any previously unmapped iCLIP reads to these exon-exon junction sequences and added any splice junction mapping reads to the total mapped dataset. After initially calling putative Ago2 bound regions by identifying clusters of more overlapping reads than a null poisson distribution model with cut-off p value < 0.001, we filtered out any clusters that had the same mismatched nucleotide in >70% of its reads. Finally, the remaining clusters were required to have significantly more tagged reads than untagged reads (binomial, p<0.05) after median-based normalization of the read counts in both libraries, using background regions found in both tagged and untagged datasets. Annotation of genomic regions was taken from Ensembl NCBIM37 build annotation. Single nucleotide 5P end files that largely reflect crosslink sites are included as BigWig files for visualization.
Supplementary_files_format_and_content: Text file including processed CLIP-seq reads and corresponding number of observations in dataset.
Submission date Apr 05, 2013
Last update date May 15, 2019
Contact name A Bhutkar
Organization name MIT
Street address 77 Massachusetts Avenue
City Cambridge
State/province MA
ZIP/Postal code 02139
Country USA
Platform ID GPL13112
Series (2)
GSE44163 Let-7 represses Nr6a1 and a mid-gestation developmental program in adult fibroblasts
GSE45828 Let-7 represses Nr6a1 and a mid-gestation developmental program in adult fibroblasts [CLIP-Seq]
SRA SRX261259
BioSample SAMN01999542

Supplementary file Size Download File type/resource 13.4 Mb (ftp)(http) BW
GSM1116239_WT_Ago2CLIP_Tagged_MSC_p0.05enriched_clusters.bed.txt.gz 1.5 Mb (ftp)(http) TXT
GSM1116239_WT_Ago2CLIP_Tagged_sequence_counts.txt.gz 63.5 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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