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Series GSE248461 Query DataSets for GSE248461
Status Public on Jun 13, 2024
Title An in situ method for identification of transcriptome-wide protein-RNA interactions in cells [in_situ_STAMP II]
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Summary RNA-binding proteins (RBPs) play important roles in RNA metabolism including splicing, stability, localization, and translation. RBP-RNA interaction profiles are indicative of many diseases. Existing methods for mapping RBP-RNA interactions transcriptome-wide have notable limitations: immunoprecipitation (IP)-based technologies require large quantities of input materials, and RNA shearing during processing prevents identification of RNA isoforms. Meanwhile, profiling methods using RNA-modifying enzymes require ectopic expression of fusion proteins in cells of interest, potentially distorting interaction profiles. Here we report in situ STAMP, an RBP-RNA profiling method that overcomes the limitations of existing methods. In situ STAMP utilizes a chimeric fusion of the cytosine deaminase APOBEC1 and an IgG-targeting single-domain antibody (nanobody). We demonstrate that this fusion protein can be specifically targeted to proteins of interest including the RBPs RBFOX2 and TDP-43 when combined with primary antibodies targeting these proteins, enabling identification of their binding sites in un-engineered HEK293T cells. The canonical binding motifs of both RBFOX2 (UGCAUG) and TDP43 (UGUGUG) could be identified by de novo motif analysis from in situ STAMP data, demonstrating the method’s high specificity. In situ STAMP preserves intact RNAs and is therefore compatible with direct cDNA PacBio long-read sequencing, enabling the method to distinguish between RNA isoforms. Importantly, in situ STAMP is compatible with multiple fixation methods including methanol and formaldehyde fixation, enabling its application to tissue samples collected in research or clinical settings. Thus, in situ STAMP enables the profiling of authentic RBP-RNA interactions using small quantities of primary cells or tissues, thereby bridging a critical gap in uncovering the roles of RBPs in RNA-related disease mechanisms in authentic biological contexts.
 
Overall design in situ STAMP (C-to-U RNA editing) in fixed cells
 
Contributor(s) Liang Q, Yu T, Kofman E, Jagannatha P, Rhine K, Corbett KD, Yeo GW
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Submission date Nov 22, 2023
Last update date Jun 14, 2024
Contact name Brian Yee
E-mail(s) brian.alan.yee@gmail.com
Organization name UCSD
Department Health
Lab Yeo
Street address 9500 Gilman Dr
City La Jolla
State/province CA
ZIP/Postal code 92093
Country USA
 
Platforms (1)
GPL30173 NextSeq 2000 (Homo sapiens)
Samples (14)
GSM7914703 100pg_MeOH_HEK293T_RBFOX2_replicate1
GSM7914704 100pg_MeOH_HEK293T_RBFOX2_replicate2
GSM7914705 100pg_MeOH_HEK293T_RBFOX2_replicate3
This SubSeries is part of SuperSeries:
GSE240326 An in situ method for identification of transcriptome-wide protein-RNA interactions in cells
Relations
BioProject PRJNA1044067

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE248461_100pg_Buffer_only_confident_peaks.bed.gz 3.7 Kb (ftp)(http) BED
GSE248461_100pg_RBFOX2_cleaned_confident_peaks.bed.gz 10.5 Kb (ftp)(http) BED
GSE248461_100pg_RBFOX2_confident_peaks.bed.gz 12.1 Kb (ftp)(http) BED
GSE248461_1ng_Buffer_only_confident_peaks.bed.gz 9.6 Kb (ftp)(http) BED
GSE248461_1ng_RBFOX2_cleaned_confident_peaks.bed.gz 37.3 Kb (ftp)(http) BED
GSE248461_1ng_RBFOX2_confident_peaks.bed.gz 41.8 Kb (ftp)(http) BED
GSE248461_RAW.tar 14.5 Mb (http)(custom) TAR (of BED, TSV)
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Raw data are available in SRA

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