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Series GSE198250 Query DataSets for GSE198250
Status Public on Jul 30, 2022
Title High depth profiling of miRNA targets with chimeric eCLIP [eCLIPseq]
Organisms Homo sapiens; Mus musculus; Rattus norvegicus
Experiment type Expression profiling by high throughput sequencing
Other
Summary Growing demand in RNA-targeted therapies and promise of miRNA-based drugs creates a need for tools that can accurately identify and quantify miRNA:target interactions at scale. The experimental capture of miRNA:mRNA interactions by ligation into chimeric RNA fragments provides a direct read out of miRNA targets by enabling profiling of miRNA targets with high-throughput sequencing. However, integration of chimeric CLIP-seq into wide practical use has been limited because the inefficiency of the miRNA:mRNA ligation step (resulting in a low rate of chimeric reads in final libraries) combined with the technical complexity of the method makes it challenging to apply to miRNAs of interest at scale. Here we describe chimeric eCLIP, in which we integrate a chimeric ligation step into AGO2 eCLIP to enable chimeric read recovery, and show that removal of the cumbersome polyacrylamide gel and nitrocellulose membrane transfer step common to CLIP techniques can be omitted for chimeric AGO2 eCLIP to create a simplified high throughput version of the assay that maintains high signal-to-noise. With the increased yield of recovered miRNA:mRNA interactions in no-gel chimeric eCLIP, we show that simple enrichment steps using either PCR or on-bead probe capture can be added to chimeric eCLIP in order to target and enrich libraries for chimeric reads specific to one or more miRNAs of interest in both cell lines and tissue samples, resulting in 30- to 175-fold increases in recovery of chimeric reads for miRNAs of interest. We further show that the same probe-capture approach can be used to recover miRNA interactions for a targeted gene of interest, revealing both distinct miRNA targeting as well as co-targeted by several miRNAs from the same seed family. RNA-seq analysis of gene expression following miRNA overexpression confirmed miRNA-mediated repression of chimeric eCLIP identified targets, and indicated that chimeric eCLIP can provide additional sensitivity to detect regulated targets among genes that either contain or lack computationally predicted miRNA target sites. Thus, we believe that chimeric eCLIP will be a useful tool for quantitative profiling of miRNA targets in varied sample types at scale, and revealing a deeper picture for regulatory networks for miRNAs of biological interest.
 
Overall design Chimeric eCLIP performed with gel and without gel in human HEK293T, mouse liver, rat C9 and HEK293T+C9 mix cells.
 
Contributor(s) Manakov SA, Yee BA, Shen K, Cox D, Park S, Shishkin AA, Yeo GW, Van Nostrand EL
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Submission date Mar 09, 2022
Last update date Aug 01, 2022
Contact name Gene Yeo
E-mail(s) geneyeo@ucsd.edu
Organization name UCSD
Street address 2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab
City La Jolla
State/province CA
ZIP/Postal code 92037
Country USA
 
Platforms (5)
GPL20301 Illumina HiSeq 4000 (Homo sapiens)
GPL24247 Illumina NovaSeq 6000 (Mus musculus)
GPL24676 Illumina NovaSeq 6000 (Homo sapiens)
Samples (188)
GSM5942087 Expt1_293T_WithGelChimeCLIP_total_rep1
GSM5942088 Expt1_293T_WithGelChimeCLIP_total_rep2
GSM5942089 Expt1_293T_NoGelChimeCLIP_total_rep1
This SubSeries is part of SuperSeries:
GSE198251 High depth profiling of miRNA targets with chimeric eCLIP
Relations
BioProject PRJNA814286

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Supplementary file Size Download File type/resource
GSE198250_RAW.tar 4.6 Gb (http)(custom) TAR (of BED, BW, TSV)
GSE198250_R_Chi_2_1g.vs.R_Chi_10_2g.bed.gz 380.2 Kb (ftp)(http) BED
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Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record
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