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| Status |
Public on Dec 01, 2014 |
| Title |
celf1_clip_heart |
| Sample type |
SRA |
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| Source name |
heart (ventricle)
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| Organism |
Mus musculus |
| Characteristics |
strain background: C57/BL6
cell/tissue type: heart (ventricle)
ip antibody: anti-cugbp1 antibody clone 3B1
ip antibody vendor: Millipore
ip antibody cat. #: 05-621
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| Extracted molecule |
total RNA |
| Extraction protocol |
CLIP was performed using 254 nm UV irradiation as previously described (PMID 19272451), using heart tissue or muscle tissue of 16-week-old mice or cultured C2C12 mouse myoblasts.
Tissue was ground to a powder using liquid nitrogen-cooled mortar and pestle prior to UV irradiation. The dry powder was placed into a 10 cm2 tissue culture dish, sitting on ice, and crosslinked 3 x 400 mJ/cm2. In between each round of cross-linking, the dish was shaken from side to side, to redistribute the tissue powder and provide maximum opportunity for all tissue particles to be cross-linked. The tissue was then lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% sodium deoxycholate, 1% NP-40, 0.5% SDS). The lysate was treated with DNase and RNase If (NEB) for 10 minutes at 37 degrees, with dilutions of 1:10,000 and 1:50,000 providing optimal RNA fragment lengths for downstream purification. Immunoprecipitation was performed using the 3B1 antibody clone against CELF1 (Millipore) and protein A beads (Invitrogen). The beads were washed twice with RIPA and twice with RIPA containing 1M NaCl. The 3' adapter was pre-adenylated with ImpA (Hafner et al., 2008) and ligated while the RNA-protein complexes were on beads using T4 RNA ligase (Rnl2 truncation) in the absence of ATP (NEB). The complexes were run on SDS-PAGE gel, transferred to nitrocellulose, and isolated from membrane as previously described (Wang et al., 2009). The protein was digested by proteinase K, and RNA was precipitated, and ligated to the 5' adapter using T4 RNA ligase (NEB). Two nucleotide-long barcode sequences were used at the 3' end of the 5' adapter, to allow for censoring of CLIP read PCR duplications (Adapter and primer sequences (5' to 3') are provided in the CLIP-seq_readme.txt). Ligated products were resolved by electrophoresis on TBE-urea polyacrylamide gels, isolated, and subjected to RT-PCR using Superscript III enzyme (Invitrogen) and Phusion polymerase (NEB). PCR primers contained indexes for sequence multiplexing, but for these experiments no samples were multiplexed.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
library strategy: CLIP-seq
Reads were trimmed of adapters, and aligned to the mm9 mouse genome and splice junctions using tophat/bowtie.
CLIP clusters were identified as described in Yeo et al, NSMB 2009. Briefly, overlapping reads were combined and tested to assess whether their density is consistent with a null model in which reads would be distributed according to a poisson distribution. These bed files contain only clusters found within Entrez gene boundaries.
Genome_build: mm9
Supplementary_files_format_and_content: Bed file of CLIP clusters
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| Submission date |
Sep 30, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Eric T Wang |
| Organization name |
University of Florida
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| Street address |
2033 Mowry Rd
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| City |
Gainesville |
| State/province |
FL |
| ZIP/Postal code |
32610 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE61892 |
Functional Antagonism Between CELF and Mbnl Proteins in Cytoplasm and Nucleus [CLIP-seq] |
| GSE61893 |
Functional Antagonism Between CELF and Mbnl Proteins in Cytoplasm and Nucleus |
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| Relations |
| BioSample |
SAMN03084649 |
| SRA |
SRX717360 |
| Named Annotation |
GSM1516651_celf1_clip_heart.clusters.bed.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1516651_celf1_clip_heart.clusters.bed.gz |
577.1 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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