|
| 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] |
|
| Relations |
| SRA |
SRX261259 |
| BioSample |
SAMN01999542 |
