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E-GEOD-62677 - MiR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction [II]

Status
Released on 22 December 2014, last updated on 2 January 2015
Organism
Homo sapiens
Samples (8)
Protocols (3)
Description
Identifying the interaction partners of non-coding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA-cross-linking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-cross-linking and Argonaute 2-immunopurification followed by streptavidin affinity-purification of probe-linked RNAs provided selectivity in the capture of targets, identified by deep-sequencing. MiR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. MiR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long-non-coding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a sponge for these miRNAs. Two replicates of four cDNA libraries were submitted to deep sequencing: an RNA “Input” sample from RNA-12-transfected cells; a sample of the Ago2-immunopurified RNA from cells treated with transfection reagent (“Mock”); a sample of the Ago2-immunopurified RNA from RNA-12-treatment (“Ago2-IP”); and the sample from miR-CLIP-purified RNA (“miR-CLIP”).
Experiment types
other, RNA-seq of coding RNA 
Contacts
Jochen Imig <geo@ncbi.nlm.nih.gov>, Andreas Brunschweiger, Anneke Brümmer
MINSEQE
Exp. designProtocolsVariablesProcessedSeq. reads
Files
Investigation descriptionE-GEOD-62677.idf.txt
Sample and data relationshipE-GEOD-62677.sdrf.txt
Additional data (1)E-GEOD-62677.additional.1.zip
Links